Developmental appearance of dentin matrix protein 1 during the early dentinogenesis in rat molars as identified by high-resolution immunocytochemistry

Dentin matrix protein 1 (DMP 1) is an acidic phosphoprotein that has been postulated to play an important role in mineralized tissue formation. We have examined rat molar tooth germs by applying a high-resolution immunocytochemical approach with the purpose to identify the temporal and spatial localization of DMP 1 at the onset of dentinogenesis. Upper molar tooth germs of 2- to 3-day-old Wistar rats were fixed in a cacodylate-buffered 0.1% glutaraldehyde + 4% formaldehyde fixative, left unosmicated and embedded in LR White resin. The sections were incubated with a polyclonal DMP 1 antibody for postembedding colloidal gold immunolabeling and examined in a Jeol 1010 transmission electron microscope. The earliest localization of DMP 1 was in the Golgi region as well as in the nucleus of differentiating odontoblasts. When mineralization spread from matrix vesicles to the surrounding matrix, DMP 1 was extracellularly detected around the mineralizing globules. In the regions of fully mineralized mantle dentin, it was present in the mineralized regions, mainly around the peritubular dentin. The appearance of DMP 1 during early dentinogenesis implies a direct role for this protein in both odontoblast differentiation and matrix mineralization.     

Modeling breast cancer in vivo and ex vivo reveals an essential role of Pin1 in tumorigenesis

Phosphorylation on certain Ser/Thr-Pro motifs is a major oncogenic mechanism. The conformation and function of phosphorylated Ser/Thr-Pro motifs are further regulated by the prolyl isomerase Pin1. Pin1 is prevalently overexpressed in human cancers and implicated in oncogenesis. However, the role of Pin1 in oncogenesis in vivo is not known. We have shown that Pin1 ablation is highly effective in preventing oncogenic Neu or Ras from inducing cyclin D1 and breast cancer in mice, although it neither affects transgene expression nor mammary gland development. Moreover, we have developed an ex vivo assay to uncover that a significant fraction of primary mammary epithelial cells from Neu or Ras mice display various malignant properties long before they develop tumors in vivo. Importantly, these early transformed properties are effectively suppressed by Pin1 deletion, which can be fully rescued by overexpression of cyclin D1. Thus, Pin1 is essential for tumorigenesis and is an attractive anticancer target. Our ex vivo assay can be used to study early events of breast cancer development in genetically predisposed mice.     

Estrogen-induced regulation of the ATP-binding cassette transporter A1 (ABCA1) in mice: A possible mechanism of atheroprotection by estrogen

Estrogens are suggested to be antiatherogenic by affecting the vessel wall components. Since ABCA1 was recently shown to be atheroprotective, it was examined if estrogen-induced atheroprotection occurs partly via the regulation of the ABCA1. Since hepatic ABCA1 expression was also suggested to contribute to the bulk HDL levels, regulation of the ABCA1 under conditions of high or low levels of HDL were investigated in mice expressing normal or elevated levels of apoAI. To delineate whether estrogen's effect occurs via estrogen receptor--mediated pathway, the estrogen receptor--deficient (ER-)^–/– mice were also administered either placebo or -estradiol for 5 consecutive days. Estrogen treatments decreased circulating HDL levels by 30%, but increased hepatic and intestinal ABCA1 mRNA by 2- and 1.5-fold, respectively. Hepatic ABCA1 mRNA also increased in the ER-^–/– mice by 3-fold. These results suggest that estrogen, despite lowering the levels of HDL, it up-regulated the hepatic ABCA1 mRNA, and in the absence of ER-, ER- could compensate for ER-. To study whether HDL levels correlate with the ABCA1 expression, wild-type (WT) and the apoAI transgenic (A1-Tg) mice were fed high fat (HF) diet with or without cholic acid (CA) for 3 weeks. One group of mice was treated with fenofibrate, known to elevate HDL levels. CA without HF decreased HDL levels, while fenofibrate increased HDL levels. However, neither CA nor fenofibrate altered hepatic ABCA1 mRNA levels. HF diet increased the hepatic ABCA1 mRNA 1.8-fold in WT, but lowered ABCA1 mRNA by 2-fold in A1-Tg mice, suggesting that ABCA1 levels did not correlate with circulating HDL levels, while basal levels of HDL influenced ABCA1 expression. These data show for the first time that estrogen's antiatherogenic effects may occur via ABCA1-mediated pathway, and circulating HDL levels may influence expression of ABCA1.     

Identification of putative in vivo substrates of calpain 3 by comparative proteomics of overexpressing transgenic and nontransgenic mice

Calpain 3 (CAPN3) is a calcium-dependent protease, mutations in which cause limb girdle muscular dystrophy type 2A. To explore the physiological function of CAPN3, we compared the proteomes of transgenic mice that overexpress CAPN3 (CAPN3 Tg) and their nontransgenic (non-Tg) counterparts. We first examined known muscular dystrophy-related proteins to determine if overexpression of CAPN3 results in a change in their distribution or concentration. This analysis did not identify any known muscular dystrophy proteins as substrates of CAPN3. Next, we used a proteomic approach to compare and identify differentially represented proteins in 2-DE of CAPN3 Tg and non-Tg mice. LC-MS/MS analysis led to the identification of ten possible substrates for CAPN3, classified into two major functional categories: metabolic and myofibrillar. Myosin light chain 1 (MLC1) was focused upon because our previous studies suggested a role for CAPN3 in sarcomere remodeling. In this study, CAPN3 was shown to proteolyze MLC1 in vitro. These studies are the first to identify possible substrates for CAPN3 in an in vivo system and support a role for CAPN3 in sarcomere remodeling by cleavage of myofibrillar proteins such as MLC1. In addition, these data also suggest a role for CAPN3 in mitochondrial protein turnover.     

Modulation of neuropeptide Y and Y1 receptor expression in the amygdala by fluctuations in the brain content of neuroactive steroids during ethanol drinking discontinuation in Y1R/LacZ transgenic mice

Previous studies have shown that GABAergic neuroactive steroids increase Y₁ receptor (Y₁R) gene expression in the amygdala of Y ₁ R / LacZ transgenic mice, harbouring the murine Y₁R gene promoter linked to a LacZ reporter gene. As ethanol is known to increase GABAergic neuroactive steroids, we investigated the relationship between fluctuations in the brain content of neuroactive steroids induced by chronic voluntary ethanol consumption or ethanol discontinuation and both the level of neuropeptide Y (NPY) immunoreactivity and Y₁R gene expression in the amygdala of Y ₁ R / LacZ transgenic mice. Ethanol discontinuation (48 h) after voluntary consumption of consecutive solutions of 3%, 6%, 10% and 20% (v/v) ethanol over 4 weeks produced an anxiety-like behaviour as measured by elevated plus maze. Voluntary ethanol intake increased the cerebrocortical concentration of the progesterone metabolite 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG) that returned to control level 48 h after discontinuation of ethanol intake. Ethanol discontinuation significantly decreased NPY immunoreactivity and concomitantly increased Y ₁ R / LacZ transgene expression in the amygdala, whereas chronic ethanol intake failed to affect these parameters. The 5α-reductase inhibitor finasteride prevented both the increase in the cerebrocortical concentration of 3α,5α-TH PROG apparent after 4 weeks of ethanol intake and the changes in NPY immunoreactivity and transgene expression induced by ethanol discontinuation. Data suggest that 3α,5α-TH PROG plays an important role in the changes in NPY–Y₁R signalling in the amygdala during ethanol discontinuation.     

Changes in expression of the neuropeptide Y Y1 receptor gene in the medial amygdala of transgenic mice during pregnancy and after delivery

Long-term administration of progesterone or allopregnanolone was previously shown to increase Y1 receptor gene expression in the medial amygdala of Y1R/LacZ transgenic mice, which harbor a construct comprising the murine Y1 receptor gene promoter and a lacZ reporter. We have now investigated the effects of physiological fluctuations in the cerebrocortical concentrations of neuroactive steroids during pregnancy on Y1R/LacZ transgene expression by quantitative histochemical analysis of beta-galactosidase activity. Cerebrocortical concentrations of progesterone and its metabolites allopregnanolone and allotetrahydrodeoxycorticosterone were increased on day 18 of pregnancy and had returned to control values 2 days after delivery. Transgene expression in the medial amygdala was also increased on day 18 of pregnancy and had returned to control values 2 days after delivery. Similar results were obtained after analysis of Y1R mRNA levels in the medial amygdala of pregnant mice by in situ hybridization. Administration of the 5alpha-reductase inhibitor finasteride to pregnant mice prevented both the increase in the cerebrocortical concentrations of neuroactive steroids as well as the increase in transgene expression. These data suggest that fluctuations in the brain concentrations of endogenous neuroactive steroids during pregnancy are associated with changes in Y1 receptor gene expression in the medial amygdala, further supporting a functional interaction between the GABAergic and NPY-Y1 receptor systems.     

Lipid peroxidation up-regulates BACE1 expression in vivo: a possible early event of amyloidogenesis in Alzheimer's disease

Increased lipid peroxidation is shown to be an early event of Alzheimer's disease (AD). However, it is not clear whether and how increased lipid peroxidation might lead to amyloidogenesis, a hallmark of AD. Glutathione peroxidase 4 (Gpx4) is an essential antioxidant defense enzyme that protects an organism against lipid peroxidation. Gpx4+/- mice show increased lipid peroxidation in brain, as evidenced by their elevated levels of 4-hydroxy-2-nonenal. To understand the role of lipid peroxidation in amyloidogenesis, we studied secretase activities in Gpx4+/- mice as a function of age. Both young (6 months) and middle-aged (17-20 months) Gpx4+/- mice had higher levels of beta-secretase activity than their age-matched wildtype controls, and the increased beta-secretase activity in Gpx4+/- mice was a result of up-regulation of beta-site amyloid precursor protein cleavage enzyme 1 (BACE1) expression at the protein level. The high level of BACE1 protein led to increased endogenous beta-amyloid (Abeta)(1-40) in middle-aged Gpx4+/- mice. We further studied amyloidogenesis in APPGpx4+/- mice. Our data indicate that APPGpx4+/- mice had significantly increased amyloid plaque burdens and increased Abeta(1-40) and Abeta(1-42) levels compared with APPGpx4+/+ mice. Therefore, our results indicate that increased lipid peroxidation leads to increased amyloidogenesis through up-regulation of BACE1 expression in vivo, a mechanism that may be important in pathogenesis of AD at early stages.     

Increased expression of the neuropeptide Y receptor Y1 gene in the medial amygdala of transgenic mice induced by long-term treatment with progesterone or allopregnanolone

The neurosteroid allopregnanolone, a reduced metabolite of progesterone, induces anxiolytic effects by enhancing GABA(A) receptor function. Neuropeptide Y (NPY) and GABA are thought to interact functionally in the amygdala, and this interaction may be important in the regulation of anxiety. By using Y(1)R/LacZ transgenic mice, which harbour a fusion construct comprising the promoter of the mouse gene for the Y(1) receptor for NPY linked to the lacZ gene, we previously showed that long-term treatment with benzodiazepine receptor ligands modulates Y(1) receptor gene expression in the medial amygdala. We have now investigated the effects of prolonged treatment with progesterone or allopregnanolone on Y(1)R/LacZ transgene expression, as determined by quantitative histochemical analysis of beta-galactosidase activity. Progesterone increased both the cerebrocortical concentration of allopregnanolone and beta-galactosidase expression in the medial amygdala. Finasteride, a 5alpha-reductase inhibitor, prevented both of these effects. Long-term administration of allopregnanolone also increased both the cortical concentration of this neurosteroid and transgene expression in the medial amygdala. Treatment with neither progesterone nor allopregnanolone affected beta-galactosidase activity in the medial habenula. These data suggest that allopregnanolone regulates Y(1) receptor gene expression through modulation of GABA(A) receptor function, and they provide further support for a functional interaction between GABA and neuropeptide Y in the amygdala.     

Vitamin D limits inflammation-linked microRNA expression in adipocytes in vitro and in vivo: A new mechanism for the regulation of inflammation by vitamin D

Inflammation of adipose tissue is believed to be a contributing factor to many chronic diseases associated with obesity. Vitamin D (VD) is now known to limit this metabolic inflammation by decreasing inflammatory marker expression and leukocyte infiltration in adipose tissue. In this study, we investigated the impact of VD on microRNA (miR) expression in inflammatory conditions in human and mouse adipocytes, using high-throughput methodology (miRNA PCR arrays). Firstly, we identified three miRs (miR-146a, miR-150, and miR-155) positively regulated by TNFα in human adipocytes. Interestingly, the expression of these miRs was strongly prevented by 1,25(OH)₂D preincubation. These results were partly confirmed in 3T3-L1 adipocytes (for miR-146a and miR-150). The ability of VD to control the expression of these miRs was confirmed in diet-induced obese mice: the levels of the three miRs were increased following high fat (HF) diet in epididymal white adipose tissue and reduced in HF diet fed mice supplemented with VD. The involvement of NF-κB signaling in the induction of these miRs was confirmed in vitro and in vivo using aP2-p65 transgenic mice. Finally, the ability of VD to deactivate NF-κB signaling, via p65 and IκB phosphorylation inhibition in murine adipocyte, was observed and could constitute a driving molecular mechanism. This study demonstrated for the first time that VD modulates the expression of miRs in adipocytes in vitro and in adipose tissue in vivo through its impact on NF-κB signaling pathway, which could represent a new mechanism of regulation of inflammation by VD.     

Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific growth factors that appear to play key roles in granulosa cell development and fertility in most mammalian species. We have evaluated the role(s) of these paracrine factors in the development and function of both the cumulus cells and oocytes by assessing cumulus expansion, oocyte maturation, fertilization, and preimplantation embryogenesis in Gdf9+/-Bmp15-/- [hereafter, double mutant (DM)] mice. We found that cumulus expansion, as well as the expression of hyaluronon synthase 2 (Has2) mRNA was impaired in DM oocyte-cumulus cell complexes. This aberrant cumulus expansion was not remedied by coculture with normal wild-type (WT) oocytes, indicating that the development and/or differentiation of cumulus cells in the DM, up to the stage of the preovulatory luteinizing hormone (LH) surge, is impaired. In addition, DM oocytes failed to enable FSH to induce cumulus expansion in WT oocytectomized (OOX) cumulus. Moreover, LH-induced oocyte meiotic resumption was significantly delayed in vivo, and this delayed resumption of meiosis was correlated with the reduced activation of mitogen-activated protein kinase (MAPK) in the cumulus cells, thus suggesting that GDF9 and BMP15 also regulate the function of cumulus cells after the preovulatory LH surge. Although spontaneous in vitro oocyte maturation occurred normally, oocyte fertilization and preimplantation embryogenesis were significantly altered in the DM, suggesting that the full complement of both GDF9 and BMP15 are essential for the development and function of oocytes. Because receptors for GDF9 and BMP15 have not yet been identified in mouse oocytes, the effects of the mutations in the Bmp15 and Gdf9 genes on oocyte development and functions must be produced indirectly by first affecting the granulosa cells and then the oocyte. Therefore, this study provides further evidence for the existence and functioning of an oocyte-granulosa cell regulatory loop.     

Prevention of autoimmune diabetes in NOD mice by troglitazone is associated with modulation of ICAM-1 expression on pancreatic islet cells and IFN-gamma expression in splenic T cells

Thiazolidinediones acting as PPAR-gamma agonists are a new generation of oral antidiabetics addressing insulin resistance as a main feature of type-2 diabetes. In accordance to our results, pre-clinical studies have demonstrated that the thiazolinedione troglitazone prevents the development of insulin-dependent autoimmune type-1 diabetes. To investigate whether TGZ acts by affecting the ICAM-1/LFA-1 pathway and/or the Th1/Th2 cytokine balance in NOD mice, we analysed the IL-1beta-induced ICAM-1 expression on islet-cells and the LFA-1, CD25, IL-2, IFN-gamma, IL-4, and IL-10 expression on splenocytes. After 200 days of oral TGZ administration, islet cells from TGZ-treated NOD mice showed a reduced ICAM-1 expression in response to the pro-inflammatory cytokine IL-1beta. The expression of the ligand LFA-1 on CD4(+) and CD8(+) T-cells was comparable to that of placebo- and untreated controls. Also, the expression of Th1/Th2 cytokines was comparable in groups receiving TGZ or Placebo. Nevertheless, the investigated NOD mice segregated into IFN-gamma low- and IFN-gamma high producers as revealed by cluster analysis. Interestingly, the majority of TGZ-treated mice belonged to the cluster of IFN-gamma low producers. Thus, the prevention of autoimmune diabetes in NOD mice by TGZ seems to be associated with suppression of IL-1beta-induced ICAM-1 expression leading to a reduced vulnerability of pancreatic beta-cells during the effector stage of beta-cell destruction. In addition, IFN-gamma production was modulated, implicating that alteration of the Th1/Th2 cytokine balance might have contributed to diabetes prevention. The findings of this study suggest that TGZ exerts its effects by influencing both the beta-cells as the target of autoimmune beta-cell destruction and the T-cells as major effectors of the autoimmune process.     

Cox-2 expression, PGE2 and cytokines production are inhibited by endogenously synthesized n-3 PUFAs in inflamed colon of fat-1 mice

There is great interest in the role of polyunsaturated fatty acids (PUFAs) in promoting (n-6 class) or inhibiting (n-3 class) inflammation. Mammalian cells are devoid of desaturase that converts n-6 to n-3 PUFAs. Consequently, essential n-3 fatty acids must be supplied with the diet. We have studied the effect of endogenously produced n-3 PUFAs on colitis development in fat-1 transgenic mice carrying the Caenorhabditis elegans fat-1 gene encoding n-3 desaturase. Colonic cell lipid profile was measured by capillary gas chromatography in fat-1 and wild-type (WT) littermates fed standard diet supplemented with 10% (w/w) safflower oil rich (76%) in n-6 polyunsaturated linoleic acid (LA). Experimental colitis was induced by administrating 3% dextran sodium sulphate (DSS). Colitis was scored by histopatological analysis. Cyclooxygenase-2 (Cox-2) expression was evaluated by real time polymerase chain reaction. Prostaglandin E₂ (PGE₂) levels and cytokine production were determined by enzyme and microsphere-based immunoassays, respectively. The n-6/n-3 PUFA ratios in colonic cells of fat-1 mice were markedly lower (9.83±2.62) compared to WT (54.5±9.24, P<.001). Results also showed an attenuation of colonic acute and chronic inflammation in fat-1 mice with significant decreases in PGE₂ production (P<.01) and Cox-2 expression (P<.01). High levels of colitis-induced proinflammatory cytokines, interleukin (IL)-18, IL-1α, IL-1β, IL-6, monocytes chemotactic proteins 1, 2 and 3 (MCP 1,2,3), matrix metalloproteinase 9 and tumor necrosis factor α (TNF-α) were down-regulated in DSS acutely and chronically treated fat-1 mice. The expression of fat-1 gene in the colon was associated with endogenous n-3 PUFAs production, decreased Cox-2 expression, increased PGE₂ and cytokine production.     

Heme oxygenase-1 and heme oxygenase-2 have distinct roles in the proliferation and survival of olfactory receptor neurons mediated by cGMP and bilirubin,respectively

Heme oxygenase (HO) is implicated in protection against oxidative stress, proliferation and apoptosis in many cell types, including neurons. We utilized olfactory receptor neurons (ORNs) as a model to define the roles of HO-1 and HO-2 in neuronal development and survival, and to determine the mediators of these effects. The olfactory system is a useful model as ORNs display neurogenesis post-natally and do not contain nitric oxide synthase (NOS) activity, which could confound results. HO isoforms were expressed in ORNs during embryogenesis and post-natally. Mice null for either HO-1 or HO-2 displayed decreased proliferation of neuronal precursors. However, apoptosis was increased only in HO-2 null mice. Cyclic GMP immunostaining was reduced in ORNs in both genotypes, providing direct evidence that HO mediates cGMP production in vivo. Bilirubin immunostaining was reduced only in HO-2 null mice. These roles for HO-1 and HO-2 were confirmed using detergent ablation of the epithelium to observe increased neurogenesis of ORNs after target disruption in HO null mice. Primary cultures of ORNs revealed that proliferative and survival effects of HO were mediated through cGMP and bilirubin, respectively. These results support a role for HO, the CO-cGMP signaling system and bilirubin in neurodevelopment and in response to injury.     

Antisense attenuation of Wnt-1 and Wnt-3a expression in whole embryo culture reveals roles for these genes in craniofacial,spinal cord,and cardiac morphogenesis

Wnt-1 and Wnt-3a proto-on-cogenes have been implicated in the development of midbrain and hindbrain structures. Evidence for such a role has been derived from in situ hybridization studies showing Wnt-1 and -3a expression in developing cranial and spinal cord regions and from studies of mutant mice whose Wnt-1 genes have undergone targeted disruption by homologous recombination. Wnt-1 null mutants exhibit cranial defects but no spinal cord abnormalities, despite expression of the gene in these regions. The absence of spinal cord abnormalities is thought to be due to a functional compensation of the Wnt-1 deficiency by related genes, a problem that has complicated the analysis of null mutants of other developmental genes as well. Herein, we describe the attenuation of Wnt-1 expression using antisense oligonucleotide inhibition in mouse embryos grown in culture. We induce similar mid- and hindbrain abnormalities as those seen in the Wnt-1 null mutant mice. Attentuation of Wnt-1 expression was also associated with cardiomegaly resulting in hemostasis. These findings are consistent with the possibility that a subset of Wnt-1 expressing cells include neural crest cells known to contribute to septation of the truncus arteriosus and to formation of the visceral arches. Antisense knockout of Wnt-3a, a gene structurely related to Wnt-1, targeted the forebrain and midbrain region, which were hy-poplastic and failed to expand, and the spinal cord, which exhibited lateral outpocketings at the level of the forelimb buds. Dual antisense knockouts of Wnt-1 and Wnt-3a targeted all brain regions leading to incomplete closure of the cranial neural folds, and an increase in the number and severity of outpocketings along the spinal cord, suggesting that these genes complement one another to produce normal patterning of the spinal cord. The short time required to assess the mutant phenotype (2 days) and the need for limited sequence information of the target gene (20-25 nu-cleotides) make this antisense oligonucleotide/ whole embryo culture system ideal for testing the importance of specific genes and their interactions in murine embryonic development. © 1993 Wiley-Liss, Inc.     

In vivo imaging of oxidative stress in the kidney of diabetic mice and its normalization by angiotensin II type 1 receptor blocker

This study was undertaken to evaluate oxidative stress in the kidney of diabetic mice by electron spin resonance (ESR) imaging technique. Oxidative stress in the kidney was evaluated as organ-specific reducing activity with the signal decay rates of carbamoyl-PROXYL probe using ESR imaging. The signal decay rates were significantly faster in corresponding image pixels of the kidneys of streptozotocin-induced diabetic mice than in those of controls. This technique further demonstrated that administration of angiotensin II type 1 receptor blocker (ARB), olmesartan (5 mg/kg), completely restored the signal decay rates in the diabetic kidneys to control values. In conclusion, this study provided for the first time the in vivo evidence for increased oxidative stress in the kidneys of diabetic mice and its normalization by ARB as evaluated by ESR imaging. This technique would be useful as a means of further elucidating the role of oxidative stress in diabetic nephropathy.