MKP-1 mediates glucocorticoid-induced ERK1/2 dephosphorylation and reduction in pancreatic beta-cell proliferation in islets from early lactating mothers

Maternal pancreatic islets undergo a robust increase of mass and proliferation during pregnancy, which allows a compensation of gestational insulin resistance. Studies have described that this adaptation switches to a low proliferative status after the delivery. The mechanisms underlying this reversal are unknown, but the action of glucocorticoids (GCs) is believed to play an important role because GCs counteract the pregnancy-like effects of PRL on isolated pancreatic islets maintained in cell culture. Here, we demonstrate that ERK1/2 phosphorylation (phospho-ERK1/2) is increased in maternal rat islets isolated on the 19th day of pregnancy. Phospho-ERK1/2 status on the 3rd day after delivery (L3) rapidly turns to values lower than that found in virgin control rats (CTL). MKP-1, a protein phosphatase able to dephosphorylate ERK1/2, is increased in islets from L3 rats. Chromatin immunoprecipitation assay revealed that binding of glucocorticoid receptor (GR) to MKP-1 promoter is also increased in islets from L3 rats. In addition, dexamethasone (DEX) reduced phospho-ERK1/2 and increased MKP-1 expression in RINm5F and MIN-6 cells. Inhibition of transduction with cycloheximide and inhibition of phosphatases with orthovanadate efficiently blocked DEX-induced downregulation of phospho-ERK1/2. In addition, specific knockdown of MKP-1 with siRNA suppressed the downregulation of phosphoERK1/2 and the reduction of proliferation induced by DEX. Altogether, our results indicate that downregulation of phospho-ERK1/2 is associated with reduction in proliferation found in islets of early lactating mothers. This mechanism is probably mediated by GC-induced MKP-1 expression.

The effect of DMSA-functionalized magnetic nanoparticles on transendothelial migration of monocytes in the murine lung via a beta(2) integrin-dependent pathway

Magnetic nanoparticles surface-functionalized with meso-2,3-dimercaptosuccinic acid (MNPs-DMSA) constitute an innovative and promising approach for tissue- and cell-targeted delivery of therapeutic drugs in the lung. Transendothelial migration of leukocytes in the lung is a side effect of endovenous administration of MNPs-DMSA. Using cytologic and phenotypic analysis of murine bronchoalveolar lavage cells, we identified monocytes/macrophages as the main subpopulation of leukocytes involved in this process. Moreover, ultrastructural analysis revealed the presence of nanoparticles inside of numerous macrophages from bronchoalveolar lavage. MNPs-DMSA at concentrations as high as 1 X 10(15) nanoparticles/mL had no toxic effects on macrophages, as evidenced by 3-(4, 5-dimethylthiazolyi-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Notably, MNPs-DMSA up-regulated the mRNA expression of E, L- and P-selectin and macrophage-1 antigen in the murine lung. Upregulation of these cell adhesion molecules was associated with an increased concentration of tumor necrosis factor-alpha in lung. Finally, the critical relevance of the beta(2) integrin-dependent pathway in leukocyte transmigration elicited by MNPs-DMSA was demonstrated by use of knockout mice. Our results characterize mechanisms of the pro-inflammatory effects of MNPs-DMSA in the lung, and identify beta(2) integrin-targeted interventions as promising strategies to reduce pulmonary side effects of MNPs-DMSA during biomedical applications. (C) 2009 Elsevier Ltd. All rights reserved.

beta-ADRENERGIC ACTIVITY PRESERVES GLUT4 PROTEIN IN GLYCOLYTIC FIBERS IN FASTING

Glucose transporter 4 (GLUT4) expression in adipose tissue decreases during fasting. In skeletal muscle, we hypothesized that GLUT4 expression might be maintained in a beta-adrenergic-dependent way to ensure energy disposal for contractile function. Herein we investigate beta-blockade or beta-stimulation effects on GLUT4 expression in oxidative (soleus) and glycolytic [extensor digitorum longus (EDL)] muscles of fasted rats. Fasting increased GLUT4 mRNA in soleus (24%) and EDL (40%) but the protein content increased only in soleus (30%). beta 1-beta 2-, and beta 1-beta 2-beta 3-blockade decreased (20-30%) GLUT4 mRNA content in both muscles, although GLUT4 protein decreased only in EDL. When mRNA and GLUT4 protein regulations were discrepant, changes in the mRNA poly(A) tail length were detected, indicating a posttranscriptional modulation of gene expression. These results show that beta-adrenergic activity regulates GLUT4 gene expression in skeletal muscle during fasting, highlighting its participation in preservation of GLUT4 protein in glycolytic muscle. Muscle Nerve 40: 847-854, 2009

Adipocyte differentiation is inhibited by melatonin through the regulation of C/EBP beta transcriptional activity

Considering that melatonin has been implicated in body weight control, this work investigated whether this effect involves the regulation of adipogenesis. 3T3-L1 preadipocytes were induced to differentiate in the absence or presence of melatonin (10(-3) m). Swiss-3T3 cells ectopically and conditionally (Tet-off system) over-expressing the 34 kDa C/EBP beta isoform (Swiss-LAP cells) were employed as a tool to assess the mechanisms of action at the molecular level. Protein markers of the adipogenic phenotype were analyzed by Western blot. At 36 hr of differentiation of 3T3-L1 preadipocytes, a reduction of PPAR gamma expression was detected followed by a further reduction, at day 4, of perilipin, aP2 and adiponectin protein expression in melatonin-treated cells. Real-time PCR analysis also showed a decrease of PPAR gamma (60%), C/EBP alpha (75%), adiponectin (30%) and aP2 (40%) mRNA expression. Finally, we transfected Swiss LAP cells with a C/EBP alpha gene promoter/reporter construct in which luciferase expression is enhanced in response to C/EBP beta activity. Culture of such transfected cells in the absence of tetracycline led to a 2.5-fold activation of the C/EBP alpha promoter. However, when treated with melatonin, the level of C/EBP alpha promoter activation by C/EBP beta was reduced by 50% (P = 0.05, n = 6). In addition, this inhibitory effect of melatonin was also reflected in the phenotype of the cells, since their capacity to accumulate lipids droplets was reduced as confirmed by the poor staining with Oil Red O. In conclusion, melatonin at a concentration of 10(-3) m works as a negative regulator of adipogenesis acting in part by inhibiting the activity of a critical adipogenic transcription factor, C/EBP beta.

Opposite effects of fasting on TGF-beta 3 and T beta RI distribution in the gastric mucosa of suckling and early weanling rats

Objective: Our aim was to evaluate the effects of a dietary regimen (suckling or early weaning) and feeding status (fed or fasted) on the distribution of transforming growth factor-beta 3 (TGF-beta 3) and TGF receptor-I (T beta RI) in the gastric epithelium of pups Methods: Wistar rats were used At 15 d, half of the pups were separated from dams and fed with hydrated powered chow On day 17, suckling and early weanling rats were subjected to fasting (17 h). Four different conditions were established. suckling fed and fasted and early weanling fed and fasted At 18 d stomachs were collected under anesthesia and were fixed in 4% formaldehyde for immunohistochemistry The number of immunostained epithelial cells per microscopic field was determined for TGF-beta 3 and T beta RI in longitudinal sections from the gastric mucosa Results: We found that during suckling, fasting reduced the number of immunolabeled cells per field of both molecules when compared with the fed group (P < 0.05), whereas in early weaning, food restriction increased TGF-beta 3 and T beta RI distributions (P < 0.05) We also observed that TGF-beta 3 and T beta RI were more concentrated in parietal cells in the upper gland in suckling pups, whereas after early weaning these were displaced to parietal and chief cells at the bottom of the gland Conclusion: Suckling and early weaning directly influence TGF-beta 3 and T beta RI distributions in the gastric epithelium in response to fasting, such that early weaning anticipates the effects observed in adult rats. Furthermore, the differential concentrations of TGF-beta 3 and T beta RI indicate that they might be important for cell proliferation events in growth control (C) 2010 Elsevier Inc. All rights reserved

Laminin-derived peptide AG73 regulates migration, invasion, and protease activity of human oral squamous cell carcinoma cells through syndecan-1 and beta 1 integrin

Squamous cell carcinoma is a prevalent head and neck tumor with high mortality. We studied the role played by laminin alpha 1 chain peptide AG73 on migration, invasion, and protease activity of cells (OSCC) from human oral squamous cell carcinoma. Immunohistochemistry and immunofluorescence analyzed expression of laminin alpha 1 chain and MMP9 in oral squamous cells carcinoma in vivo and in vitro. Migratory activity of AG73-treated OSCC cells was investigated by monolayer wound assays and in chemotaxis chambers. AG73-induced invasion was assessed in Boyden chambers. Invasion depends on MMPs. Conditioned media from cells grown on AG73 was subjected to zymography. We searched for AG73 receptors related to these activities in OSCC cells. Immunofluorescence analyzed AG73induced colocalization of syndecan-1 and beta 1 integrin. Cells had these receptors silenced by siRNA, followed by treatment with AG73 and analysis of migration, invasion, and protease activity. Oral squamous cell carcinoma expresses laminin alpha 1 chain and MMP9. OSCC cells treated with AG73 showed increased migration, invasion, and protease activity. AG73 induced colocalization of syndecan-1 and beta 1 integrin. Knockdown of these receptors decreased AG73-dependent migration, invasion, and protease activity. Syndecan-1 and beta 1 integrin signaling downstream of AG73 regulate migration, invasion, and MMP production by OSCC cells.

NEUREGULINS 1-ALPHA AND 1-BETA ON THE REGENERATION THE PERIPHERAL NERVES

Objective: To evaluate the effect of the neuregulins 1-alpha and 1-beta on the regeneration the sciatic nerves of male adult C57BL/6J mice, using the tubulization technique. Methods: Eighteen animals were used, divided into three groups. A polyethylene prosthesis was implanted in a 4.0 mm defect of the left sciatic nerve, as follows: group 1 containing only purified collagen (Vitrogen (R)); group 2, collagen with neuregulin 1-alpha; group 3, collagen with neuregulin 1-beta. The control group consisted of six segments of right sciatic nerves. After four weeks, the animals were sacrificed. A segment from the midpoint of the nerve regenerated inside the prostheses was extracted; histological sections were standardized, and slides were made up for histomorphometric analysis. Results: the results were statistically compared using the Tukey multiple comparisons test and The Student`s t test. The animals treated with neuregulins had greater numbers of myelinized axons, with a statistically significant difference in relation to the collagen-only group. There was no statistical difference between the neuregulin 1-alpha and 1-beta groups. Conclusion: The addition of neuregulins provided a significant increase in the number of myelinized fibers.

In vivo effects of TGF beta 1 on the the growth of gastric epithelium in suckling rats

As the content of Transforming Growth Factor-beta (TGF beta) wanes in the milk of lactating rat, an increase in TGF beta is observed in the gastric epithelia concomitant with differentiation of the glands upon weaning. Whereas TGF beta has been shown to inhibit the proliferation of gastrointestinal cells in vitro, its functional significance and mechanisms of action have not been studied in vivo. Therefore, we administered TGF beta 1 (1 ng/g body wt.) to 14-day-old rats in which the gastric epithelium was induced to proliferate by fasting, and determined the involvement of signaling through Smads and the impact on epithelial cell proliferation and apoptosis. After the gavage, we observed the progressive increase of active TGF beta 1 while T beta RII-receptor remained constant in the gastric mucosa. By immunohistochemistry, we showed Smad2/3 increase at 60 min (p < 0.05) and Smad2 phosphorylation/activation and translocation to the nucleus most prominently between 0 and 30 min after treatment (p < 0.05). Importantly, TGF beta 1 inhibited cell proliferation (p < 0.05), which was estimated by BrDU pulse-labeling 12 h after gavage. Lower proliferation was reflected by increased p27(kip1) at 2 h (p < 0.05). Also, TGF beta 1 increased apoptosis as measured by M30 labeling at 60 and 180 min (p < 0.001), and by morphological features at 12 h (p < 0.05). In addition, we observed higher levels of activated caspase 3 (17 kDa) from 0 to 30 min. Altogether, these data indicate a direct effect of TGF beta 1 signaling through Smads on both inhibiting proliferation, through alteration of cycle proteins, and inducing apoptosis of gastric epithelial cells in vivo. Further, the studies suggest a potential role for both milk and tissue-expressed TG beta 1 in gastric growth during postnatal development, (C) 2007 Elsevier B.V. All rights reserved.