Giovanna Menghi

Reactivity of peroxidase-labeled lectins in rabbit submandibular and sublingual glands.

The carbohydrate histochemistry of rabbit submandibular and sublingual glands has been examined by the use of 4 peroxidase-labeled lectins at the light microscopic level. In the submandibular gland, the striated ducts appeared to be the formations which were more reactive to all lectins. In the sublingual gland, the terminal tracts were the most reactive secreting portions, because they bound all the lectins used. The material contained in the lumen of the ducts of submandibular and sublingual glands always reacted fairly intensely. The binding of lectins to salivary glands indicated the possibility to use lectins for the explanation of the transport properties both of the striated ducts and of the terminal tracts.

Involvement of p53 in Phthalate Effects on Mouse and Rat Osteoblasts

The role of two estrogen‐mimicking compounds in regulating osteoblast activities were examined. Previously, our attention was focused on benzyl butyl phthalate (BBP) and di‐n‐butyl phthalate (DBP) since previous works showed that they enter the cytoplasm, bioaccumulate, modify actin cytoarchitecture and exert mitogenic effects involving microfilament disruption, and nuclear actin and lamin A regulation in Py1a rat osteoblasts. In this study we showed that BBP and DBP cause DNA base lesions both in MT3T3‐E1 osteoblasts and in mouse primary calvarial osteoblasts (COBs). In addition, treatment with the above effectors caused an increase of p53 and phospho‐p53 (ser‐15 and ser‐20) as well as an increase of apoptotic proteins with consequent decrease of cell viability. Moreover, treatment with phthalates did not modified p53 and phospho‐p53 expression in Py1a rat osteoblasts. It is of relevance that in p53 knockdown mouse osteoblasts a proliferative effect of phthalates, similar to that observed in rat Py1a osteoblasts, was found. In conclusion, our data demonstrated that phthalates induce osteoblast apoptosis, which is, at least in part, mediated by p53 activation, suggesting that the proliferative effects could be due to p53 missing activation or p53 mutation. J. Cell. Biochem. 107: 316–327, 2009.

Prostaglandins differently regulate FGF-2 and FGF receptor expression and induce nuclear translocation in osteoblasts via MAPK kinase

We have previously reported that prostaglandin F2α (PGF2α) and its selective agonist fluprostenol increase basic fibroblast growth factor (FGF-2) mRNA and protein production in osteoblastic Py1a cells. The present report extends our previous studies by showing that Py1a cells express FGF receptor-2 (FGFR2) and that treatment with PGF2α or fluprostenol decreases FGFR2 mRNA. We have used confocal and electron microscopy to show that, under PGF2α stimulation, FGF-2 and FGFR2 proteins accumulate near the nuclear envelope and colocalize in the nucleus of Py1a cells. Pre-treatment with cycloheximide blocks nuclear labelling for FGF-2 in response to PGF2α. Treatment with SU5402 does not block prostaglandin-mediated nuclear internalization of FGF-2 or FGFR2. Various effectors have been used to investigate the signal transduction pathway. In particular, pre-treatment with phorbol 12-myristate 13-acetate (PMA) prevents the nuclear accumulation of FGF-2 and FGFR2 in response to PGF2α. Similar results are obtained by pre-treatment with the protein kinase C (PKC) inhibitor H-7. In addition, cells treated with PGF2α exhibit increased nuclear labelling for the mitogen-activated protein kinase (MAPK), p44/ERK2. Pre-treatment with PMA blocks prostaglandin-induced ERK2 nuclear labelling, as confirmed by Western blot analysis. We conclude that PGF2α stimulates nuclear translocation of FGF-2 and FGFR2 by a PKC-dependent pathway; we also suggest an involvement of MAPK/ERK2 in this process.

Enzymatic degradation and quantitative lectin labeling for characterizing glycoconjugates which act as lectin acceptors in cat submandibular gland.

SummarySites of binding of eight different lectins (LTA, UEA I, WGA, SBA, DBA, CON A, PNA, RCA I) to cat submandibular gland were studied after exposure of tissue sections to sialidase, α-fucosidase, β-galactosidase, α-mannosidase, β-N-acetylglucosaminidase. All lectins were affected by enzymatic predigestion and the labeling of individual lectins was highly dependent upon the glycosidase used to pretreat the sections. Glycoconjugates of demilunar, acinar and ductal cells exhibited a different composition of terminal sequences. For example, fucose proved to form the disaccharide fucose-galactose in demilunar and acinar cells, whereas it was present with the sequence fucose-N-acetyl-d-glucosamine in striated duct cells. Sialic acid participated both to the terminal sequence sialic acid-galactose and sialic acid-N-acetyl-d-galactosamine either in demilunar or in ductal cells. Lectin labeling combined with glycosidase digestion was also helpful in verifying the influence of neighbouring oligosaccharides on the affinity of lectins for the respective sugars.

Anti-apoptotic Bcl-2 enhancing requires FGF-2/FGF receptor 1 binding in mouse osteoblasts.

In this study, we investigated the role of prostaglandin F2α (PGF2α) in mouse osteoblast survival and the function of fibroblast growth factor 2 (FGF‐2) and fibroblast growth factor receptor 1 (FGFR1) in this process. In particular, for the first time, we demonstrated that PGF2α increased osteoblast survival in a dose‐dependent manner and we showed that the effect is correlated with an increase in Bcl‐2/Bax ratio. Furthermore, we demonstrated that PGF2α caused a decrement of the active caspases 9 and 3. By blocking FGF‐2 with the specific neutralizing antibody and by depletion of FGFR1 gene with a specific siRNA, we showed that FGFR1 and FGF‐2 are critical for the increment of Bcl‐2/Bax ratio and the decrement of the active caspases 9 and 3, induced by PGF2α. Moreover, transmission electron microscopy studies showed that PGF2α increased binding of FGF‐2 and FGFR1 and co‐localization of reactive sites at plasma membrane level. In conclusion, we report a novel mechanism in which PGF2α induces FGF‐2 binding to its specific cell surface receptor 1 leading to a cascade pathway that culminates with increased mouse osteoblast survival. J. Cell. Physiol. 214:145–152, 2008.

Signaling pathways implicated in PGF2α effects on Fgf2+/+ and Fgf2−/− osteoblasts

Prostaglandin F2α (PGF2α) regulates fibroblast growth factor‐2 (FGF‐2) and fibroblast growth factor receptor (FGFR) expression in osteoblasts. Here, the role of FGF‐2 in PGF2α‐induced proliferation and the signaling pathway involved, were determined in calvarial osteoblasts (COBs) from Fgf2+/+ and Fgf2−/− mice. The involvement of the exported FGF‐2 isoform, was determined using the FGF‐2 neutralizing antibody to alter its binding to FGFR1. PGF2α increased activity of Ras, and MAP‐kinase cascade as well as Bcl‐2 and c‐Myc levels in Fgf2+/+ but not in Fgf2−/− COBs. Moreover, in Fgf2+/+ COBs, PGF2α‐enhanced nuclear accumulation and co‐localization of Bcl‐2/c‐Myc. Although up‐regulation of multiple proliferative and survival signals were induced by PGF2α in Fgf2+/+ COBs, phospho‐p53 was unmodified while p53 was increased. Increased phospho‐p53 was, instead, found in Fgf2−/− COBs without up‐regulation of oncogenic proteins. The lack of p53 activation in wild type osteoblasts could be due in part to the overexpression of MDM2 caused by PGF2α via FGF‐2. PGF2α, also, increased cyclins D and E in Fgf2+/+ COBs and induced an expansion of Fgf2+/+ osteoblasts in G2/M phase. These data clearly show that PGF2α induces proliferation via endogenous FGF‐2 and the exported isoform mediates PGF2α effects by acting in autocrine manner. Furthermore, silencing of FGFR1 in Fgf2+/+ COBs blocked PGF2α induced increase of phospho‐MDM2 and cyclins. J. Cell. Physiol. 224: 465–474, 2010.

Sialoglycoderivatives of bovine submandibular gland identified in situ by histochemical techniques combined with lectins

SummaryWe employed sialidase procedures followed by lectin stainings combined with oxidizing and deacetylating agents to visualize the distribution and sequentiate sialoglycoconjugates in the bovine submandibular gland. In particular we evidenced in acinar and ductal cells the dishomogeneous presence of sialic acids acetylated in the polyhydroxy side chain (C7, C8, C9), whereas O-acetyl substituents at position C1 and/or C4 were not found. Sialoglycoderivatives were also differentiated by the occurrence of penultimate sugars; indeed the dimers sialic acid-(α2→3,6)-β-galactose and sialic acid-(α2→6)-α-N-acetylgalactosamine were identified. Using such technique we supported further the possibility to develop methods for the identification of the positions of Oacetyl groups and the reconstruction of terminal disaccharides within surface and cytoplasm glycoconjugates.

PGF2α increases FGF‐2 and FGFR2 trafficking in Py1a rat osteoblasts via clathrin independent and importin β dependent pathway

Previous studies showed that prostaglandin F2α (PGF2α) stimulated fibroblast growth factor‐2 (FGF‐2) and fibroblast growth factor receptor 2 (FGFR2) cytosolic and nuclear accumulation, however, the endocytic pathway has not been elucidated. This study demonstrates that although PGF2α increased the formation of clathrin‐coated structures in Py1a rat osteoblasts, they were not involved in FGF‐2 and FGFR2 trafficking. PGF2α increased binding of FGF‐2 and FGFR2 and co‐localization of reactive sites in addition to nuclear translocation at the nuclear pore complex level. FGF‐2 and FGFR2 were in close spatial correlation with importin β, further supporting nuclear import of the FGF‐2/FGFR2 complex. Immunogold and immunofluorescence techniques as well as Western blotting demonstrated increased importin β protein labeling in response to PGF2α. Similar to PGF2α, phorbol 12‐myristate 13‐acetate (PMA) also increased importin β protein. These data strongly suggest that prostaglandins may regulate osteoblast metabolism via FGF‐2/FGFR2/importin β nuclear trafficking. J. Cell. Biochem. 97: 1379–1392, 2006.

Benzyl butyl phthalate influences actin distribution and cell proliferation in rat Py1a osteoblasts.

We previously reported that transient administration of phthalates induced actin cytoskeleton disruption in Py1a osteoblasts. However, the mechanism of this transient effect was not elucidated. In this study we provided evidence that the actin cytoskeletal re‐established conditions are dependent on new actin expression and synthesis. To assess the role of phthalates in modulating the distribution of actin, confocal and electron microscopy studies were carried out. Results indicated a modification of actin distribution after phthalate administration. In addition, a relation with the nucleoskeletal component lamin A supports the hypothesis that phthalates may participate in regulatory cell processes involving actin in Py1a osteoblasts. The present study also supports the mitogenic effects of phthalates, which involve microfilament disruption, nuclear actin and lamin A. In particular, the increased levels of cyclin D3, which in mammalian cells plays a critical role in G1 to S transition and is a putative proto‐oncogene in benzyl butyl phthalate treated cells, suggested a possible effect of the endocrine disruptor in cancer processes. J. Cell. Biochem. 101: 543–551, 2007.

Specialised cell types in the chorioallantoic membrane express carbonic anhydrase during chick embryogenesis

The expression of carbonic anhydrase in the chorioallantoic membrane (CAM) of the chick embryo was investigated by means of the histochemical localisation of the enzyme catalytic sites and the immunohistochemical identification of its isoenzymatic forms. The results show that carbonic anhydrase is developmentally expressed in a subset of cells both in the ectodermal and the endodermal epithelium. The distribution patterns from both methodological approaches indicated that carbonic anhydrase is a marker of the villus cavity cells and the mitochondria‐rich cells in the ectodermal and the endodermal epithelium, respectively. Such a cell‐specific pattern of the enzyme expression provides a further contribution to characterising the heterogeneous cell population of the chick CAM and supports specific functional involvement for the distinct cell types in CAM‐mediated processes, such as calcium transport, maintenance of acid‐base balance and water and electrolyte reabsorption, during chick embryogenesis.