Luigi Marchetti

Contributions of intercalated duct cells to the normal parenchyma of submandibular glands of adult rats

The parenchyma of the submandibular gland in the adult male rat is self‐renewing, with most newly formed acinar and granular duct cells believed to differentiate from the rapidly proliferating intercalated duct (ID) compartment. Since the ID cells are phenotypically diverse, based on their different expression of perinatal secretory proteins, we systemically injected tritiated thymidine for 24 hours, and followed the pattern of thymidine distribution in cells by autoradiography and immunocytochemistry of defined cellular phenotypes over a 1‐month chase period. Proliferating cells were found within all parenchymal cell compartments; they were most numerous in ID, and primarily in those cells lacking immunoreactivity for the perinatal proteins SMG‐B1, ‐C, and ‐D. The labeling index (LI) of the ID cells reached a peak at 7 days postinjection, and then decreased over the next 3 weeks. Concurrently, the LI increased significantly in those cells at the junctions of ID with both acini and granular ducts, and also within these larger parenchymal elements. We conclude that the ID cells not reactive for perinatal proteins proliferate to expand the ID compartment, and that ID cells at the ends of the ducts differentiate into both acinar and granular duct cells. Our data provide no evidence for the differentiation of ID cells into cells of striated ducts (SD); however, the small number of excretory duct (ED) profiles seen in our preparations showed extremely high LI (>25%), suggesting that more extensive data might reveal a precursor role for the ED in replacement of SD cells. In addition to the stepwise passage of cells from ID to other parenchymal elements at their junctions, the reported occurrence of occasional clusters of B1‐positive acini (BAC) among the typical B1‐negative acini had suggested an alternate pathway, in which entire segments of newly expanded ID might develop directly into a recapitulated perinatal stage of B1‐reactive cell, pursuant to becoming mature acinar cells. Consistent with this suggestion, the BAC had a fourfold greater LI than typical adult acini; moreover, when analyzed by electron microscopic immunocytochemistry, they appeared similar to the novel perinatal Type III cells both ultrastructurally and in their pattern of B1‐immunogold labeling. In contrast, the less common acini showing a sublingual gland phenotype had no significant difference in LI from typical acinar cells. Overall, our results emphasize the importance of the nonimmunoreactive ID cells in normal cellular replacement, and the possibility that ID can undergo en bloc differentiation into replacement acini as well as incremental addition of single cells at the boundaries of ID with acini and with granular ducts. Anat Rec 263:202–214, 2001.

Endogenous FGF-2 is critically important in PTH anabolic effects on bone.

Parathyroid hormone (PTH) increases fibroblast growth factor receptor‐1 (FGFR1) and fibroblast growth factor‐2 (FGF‐2) expression in osteoblasts and the anabolic response to PTH is reduced in Fgf2−/− mice. This study examined whether candidate factors implicated in the anabolic response to PTH were modulated in Fgf2−/− osteoblasts. PTH increased Runx‐2 protein expression in Fgf2+/+ but not Fgf2−/− osteoblasts. By immunocytochemistry, PTH treatment induced nuclear accumulation of Runx‐2 only in Fgf2+/+ osteoblasts. PTH and FGF‐2 regulate Runx‐2 via activation of the cAMP response element binding proteins (CREBs). Western blot time course studies showed that PTH increased phospho‐CREB within 15 min that was sustained for 24 h in Fgf2+/+ but had no effect in Fgf2−/− osteoblasts. Silencing of FGF‐2 in Fgf2+/+ osteoblasts blocked the stimulatory effect of PTH on Runx‐2 and CREBs phosphorylation. Studies of the effects of PTH on proteins involved in osteoblast precursor proliferation and apoptosis showed that PTH increased cyclinD1‐cdk4/6 protein in Fgf2+/+ but not Fgf2−/− osteoblasts. Interestingly, PTH increased the cell cycle inhibitor p21/waf1 in Fgf2−/− osteoblasts. PTH increased Bcl‐2/Bax protein ratio in Fgf2+/+ but not Fgf2−/− osteoblasts. In addition PTH increased cell viability in Fgf2+/+ but not Fgf2−/− osteoblasts. These data suggest that endogenous FGF‐2 is important in PTH effects on osteoblast proliferation, differentiation, and apoptosis. Reduced expression of these factors may contribute to the reduced anabolic response to PTH in the Fgf2−/− mice. Our results strongly indicate that the anabolic PTH effect is dependent in part on FGF‐2 expression. J. Cell. Physiol. 219: 143–151, 2009.

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.

FGF-2 enhances Runx-2/Smads nuclear localization in BMP-2 canonical signaling in osteoblasts.

Bone morphogenetic protein 2 (BMP‐2) is one of the most potent regulators of osteoblast differentiation and bone formation. R‐Smads (Smads 1/5/8) are the major transducers for BMPs receptors and, once activated, they are translocated in the nucleus regulating transcription target genes by interacting with various transcription factors. Runx‐2 proteins have been shown to interact through their C‐terminal segment with Smads and this interaction is required for in vivo osteogenesis. In particular, recruitment of Smads to intranuclear sites is Runx‐2 dependent, and Runx‐2 factor may accommodate the dynamic targeting of signal transducer to active transcription sites. Previously, we have shown, by in vitro and in vivo experiments, that BMP‐2 up‐regulated FGF‐2 which is important for the maximal responses of BMP‐2 in bone. In this study, we found that endogenous FGF2 is necessary for BMP‐2 induced nuclear accumulation and co‐localization of Runx‐2 and phospho‐Smads1/5/8, while Runx/Smads nuclear accumulation and co‐localization was reduced in Fgf2−/− osteoblasts. Based on these novel data, we conclude that the impaired nuclear accumulation of Runx‐2 in Fgf2−/− osteoblasts reduces R‐Smads sub‐nuclear targeting with a consequent decreased expression of differentiating markers and impaired bone formation in Fgf2 null mice. J. Cell. Physiol. 228: 2149–2158, 2013.

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.

Prostaglandin F2α: A bone remodeling mediator

Prostaglandin F2α (PGF2α) plays multiple roles on bone metabolism by regulating a wide range of signaling pathways. PGF2α, via activation of PKC, stimulates Na‐dependent inorganic phosphate (Pi) transport system in osteoblasts; up‐regulates interleukin (IL)‐6 synthesis; increases vascular endothelial growth factor (VEGF). In addition, PGF2α acts as a strong mitogenic and survival agent on osteoblasts, and these effects are, at least in part, mediated by the binding of fibroblast growth factor‐2 (FGF‐2) to the specific receptor FGFR1. The understanding of PGF2α intracellular network, albeit complex to clarify, provides molecular bases useful to identify the players of osteoblast proliferation, apoptosis, and the associated angiogenic processes. Indeed, the molecular mechanism that underline PGF2α‐regulated bone metabolism may be a promising platform for the development of novel targeted therapies in the treatment of bone disorders and disease. J. Cell. Physiol. 228: 25–29, 2013.

Cell death during development of intercalated ducts in the rat submandibular gland

Programmed cell death, or apoptosis, occurs during the development of many tissues and organs in almost all multicellular organisms. Although apoptosis of salivary gland cells has been demonstrated in several pathological conditions, the role of apoptosis in the postnatal development of the salivary glands is unknown. We have studied the development of the rat submandibular gland (SMG) during its transition from the perinatal stage to the mature adult stage. Terminal tubule or Type I cells, which synthesize the secretory protein SMG‐C, are prominent in the perinatal acini and are believed to form the intercalated ducts of the adult gland. Between 25 days and 30 days after birth, the number of Type I cells and their SMG‐C immunoreactivity markedly decreased. Apoptotic cells in association with the developing intercalated ducts were labeled with the Terminal Deoxyribonucleotidyl Transferase‐Mediated dUTP Nick End Labeling (TUNEL) method. Between 25 and 40 days of age, from 50 to 80% of the apoptotic cells in cryostat sections of the SMG were closely associated with the intercalated ducts. Electron microscopy showed that the Type I cells became vacuolated, their secretory granules were reduced in size and number, and the amount of rough endoplasmic reticulum was decreased. Cellular debris resembling apoptotic bodies was phagocytosed by macrophages and adjacent intercalated duct cells. These observations suggest that the loss of Type I cells and reduction of SMG‐C immunoreactivity during development of the intercalated ducts of the adult rat SMG is due, at least in part, to apoptosis. Anat Rec 258:349–358, 2000.

Endocrine disruptors and bone metabolism.

Bone microenvironment is a complex dynamic equilibrium between osteoclasts and osteoblasts and is modulated by a wide variety of hormones and osteocyte mediators secreted in response to physiological and pathological conditions. The rate of remodeling involves tight coupling and regulation of both cells population and is regulated by a wide variety of hormones and mediators such as parathyroid hormone, prostaglandins, thyroid hormone, sex steroids, etc. It is also well documented that bone formation is easily influenced by the exposure of osteoblasts and osteoclasts to chemical compounds. Currently, humans and wildlife animals are exposed to various environmental xenoestrogens typically at low doses. These compounds, known as endocrine disruptor chemicals (EDCs), can alter the systemic hormonal regulation of the bone remodeling process and the skeletal formation. This review highlights the effects of the EDCs on mammalian bone turnover and development providing a macro and molecular view of their action.

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.