Different Expression and Localization of Phosphoinositide Specific Phospholipases C in Human Osteoblasts, Osteosarcoma Cell Lines, Ewing Sarcoma and Synovial Sarcoma

Background

Calcium plays a key role within cells, and it is involved in many signal transduction pathways, acting as a second messenger or as an enzyme cofactor. Extracellular calcium is also crucial for excitable cell membranes activity, as well as proper bone formation. Bone hardness and strength depend on mineralization, which involves a complex process in which bone-forming cells produce crystals of calcium phosphate, which were shed around the fibrous matrix. This process is strictly regulated. In addition, many signal transduction systems were interested in calcium metabolism, such as phosphoinositide (PI) pathway and related phospholipase C (PLC) enzymes. In mammals, bones represent the major calcium storage, and its capture from or release in the blood is regulated by a number of molecules, including hormones and vitamins.

Functional studies demonstrated that increase of calcium levels activates PLC, resulting in calcium increase (1).PLC cleaves phosphatydil inositol (4,5) bisphosphate (PIP2), a phosphorylated derivative of phosphatydil inositol standing in the inner half of the plasma membrane (2-7),into inositol trisphosphate (IP3) and diacylglycerol (DAG), 2 further signaling molecules. In mammalian species, the PLC family comprises modular, multi-domain enzymes covering a wide network, including direct binding to G protein subunits, small GTPases, tyrosine kinases and membrane lipids (8-10).PLC enzymes are thirteen isoenzymes subdivided into 6 subfamilies on the basis of amino acid sequence, domain structure and recruitment: β(1–4), γ(1-2), δ(1,3,4), ε(1), ζ(1), and η(1-2) (9,10). Our previous studies suggested that selected PLC enzymes are involved in osteosarcoma progression (11-25), and probably networking ezrin, a molecule acting during the metastatic spread (26-31). However, the tumor matrix mineralization of osteosarcoma was not completely highlighted (32).

In the present experiment, we analysed the PLC enzymes in different bone-related cells in order to compare expression panels. We analysed osteoblasts, human cultured osteosarcoma cell lines MG-63 and SaOS-2. We compared the obtained results to the expression of PLCs in few samples of patients affected with Ewing sarcoma (ES) and synovial sarcoma (SS).

Osteoblasts secrete the matrix components in the osseous tissue, playing a crucial role in bone formation and mineralization (33-35).

Both MG-63 (36-38) and SaOS-2 (39-41) human osteosarcoma cell lines used to be considered osteoblast-like cells, as they are currently used as osteoblastic experimental models.

The ES is the second most common primary malignant bone tumour in children and adolescents (42-45).Up to 70% of ESs arises in bone. ES is a heterogeneous family of tumours. Integration of clinical, radiological, immunohistochemical, and molecular data allow the definition of ES, although the diagnosis may result in difficult tumours showing atypical histologic features. ES is characterized by well defined genetic abnormalities, among which the most frequently represented is a translocation involving the chromosome 22 resulting in different fusion genes. The definition of histologic features is important in order to diagnose ES, although the identification of the histologic subtype does not seem to be so crucial, as shared genetic abnormalities characterize the Ewing family of tumors (46-50).

Synovial sarcoma high-grade soft tissue cancer is characterized by local invasiveness and proneness to metastasization. SS affects pediatric, adolescent, and adult population. Despite the name, probably SS arises from primitive mesenchymal cells. The genetic alteration observed up to 95% of cases and considered a specific molecular marker involves chromosome X and chromosome 18 resulting in different fusion genes (SYT-SSX1 or SYT-SSX2) (52-57).The histologic SS subtype does not assume prognostic significance (42,43).

Objectives

Our aim was to search for common patterns of expression in osteoblasts, as well as in ES and SS in order to verify differences or similarities that might result in gaining helpful elements for diagnosis and/or possible molecular therapy targets.

Methods

RNA extraction from human cell lines experiments: we analyzed cultured human osteoblasts and two osteosarcoma cell lines. MG-63 and SaOS-2 obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The initial seeding number was 250 000 cells for each experiment; cells were grown up to 1×10⁶ for molecular biology experiments. Cells were cultured as previously described (26). Briefly, cells were grown under subconfluent or confluent conditions in medium, at 37°C with 5% of CO₂. Cells were cultured in Dulbecco’s minimum essential medium (Sigma) supplemented with 10% fetal bovine serum (GIBCO) with penicillin (100 μg/mL), streptomycin (100 U/mL) and sodium pyruvate. Cells were grown for 24 hours, reaching a confluence of around 40%-60%. After the confluent monolayer was obtained, cells were detached and suspended in TRIzol reagent (Invitrogen Corporation, Carlsbad, CA). Total RNA was isolated following the manufacturer’s instructions. The purity of the RNA was assessed using a UV/visible spectrophotometer (SmartSpec 3000, Bio-Rad Laboratories, Hercules, California, USA).

RNA extraction from ES and SS samples experiments: sample biopsies of 5 patients affected with ES and of 5 patients affected with SS were analyzed. The clinical and radiological diagnosis of ES was confirmed by immunotypization with CD99, FLI1 or ERG, and genetic characterization. About 60% of the patients bore the EWS-FLI1 translocation and 40% the EWS-ERG translocation. The clinical and radiological diagnosis of SS was confirmed by histological features and genetic analyses. The sample biopsies were obtained from tumours bearing the SYT-SSX translocation. 6- to 10-um sections from formalin-fixed, paraffin-embedded specimens were used to extract mRNA using the RecoverAll™ Total Nucleic Acid Isolation kit for FFPE RNA isolation kit (Ambion Inc., Austin, TX) following the manufacturer’s instructions. Briefly, samples were deparaffined with proteinase K, homogenized and incubated overnight at 55°C. RNA was purified by the addition of RNA extraction buffer. Chloroform was then added, followed by additional incubation and centrifugation. The aqueous phase was removed to fresh tubes and the RNA was precipitated, air-dried and resuspended in 10 μL of RNA storage solution. To remove genomic DNA, all samples underwent DNase treatment. All reagents were purchased from Promega (Promega, Madison, WI, USA). The concentration and quality of the RNA obtained was monitored using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Inc. USA).

One microgram total RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription (RT) kit (Applied Byosystems, Carlsbad, California, USA) according to manufacturer’s instructions. Briefly, RT buffer, dNTP mix, RT random primers, multiscribe reverse transcriptase, RNase inhibitor and DEPC-treated distilled water were added in RNase-free tubes on ice. The RNA sample was added. The thermal cycler was programmed as follows: 25°C for 10 minutes; 37°C for 120 minutes; the reaction was stopped at 85°C for 5 minutes. The final volume was 20 µL. For polymerase chain reaction (PCR) reactions, the primer pairs (Bio Basic Inc, Amherst, New York, USA) were listed in Table 1. To amplify glyceraldehyde 3 phosphate dehydrogenase (GAPDH) (Bio Basic Inc, Amherst, New York, USA), used a constitutive positive control, the following primer pair was used: forward 5′ -CGAGATCCCTCCAAAATCAA-3′ reverse 5′-GTCTTCTGGGTGGCAGTGAT-3′. The specificity of the primers was verified by searching in the NCBI database for possible homology to cDNAs of unrelated proteins. The specificity of the primers was verified by searching in the NCBI database for possible homology to cDNAs of unrelated proteins. Each PCR tube contained the following reagents: 0.2 µM of both sense and antisense primers, 1-3 µL (about 1 µg) template cDNA, 0.2 mM dNTP mix, 2.5 U REDTaq genomic DNA polymerase (Sigma-Aldrich) and 1X reaction buffer. MgCl2 was added at a variable final concentration (empirical determination by setting the experiment). The final volume was 50 µL. The amplification was started with an initial denaturation step at 94°C for 2 minutes and was followed by 35 cycles consisting of denaturation (30 seconds) at 94°C, annealing (30 seconds) at the appropriate temperature for each primer pair and extension (1 minutes) at 72°C. The PCR products were analysed by 1.5% TAE ethidium bromide-stained agarose gel electrophoresis (Agarose Gel Unit, Bio-Rad Laboratories S.r.l., Segrate, IT). A PC-assisted CCD camera UVB lamp (Vilber Lourmat, Marne-la-Vallé France) was used for gel documentation. Gel electrophoresis of the amplification products revealed single DNA bands with nucleotide lengths as expected for each primer pair. RNA samples were also amplified by PCR without RT. No band was observed, excluding DNA contamination during the procedure (data not shown). The reaction products were further quantified with the Agilent 2100 bioanalyzer using the DNA 1000 LabChip kit (Agilent Technologies, Deutschland GmbH).

Results

Osteoblasts: PLCB1, PLCB3, PLCB4, PLCG1, PLCG2, PLCD1, PLCD3, PLCD4 and PLCE were expressed. PLCB2 was not expressed. PLCH1 and PLCH2 were inconstantly expressed.

MG-63: PLCB1, PLCB3, PLCB4, PLCG1, PLCG2, PLCD1, PLCD3, PLCE were expressed. PLCB2 was inconstantly expressed. PLCD4, PLCH1 and PLCH2 were not expressed.

SaOS-2: PLCB1, PLCB3, PLCB4, PLCG1, PLCG2, PLCD1, PLCD3, PLCE and PLCH1 were expressed. PLCB2, PLCD4 and PLCH2 were not expressed.

ES samples: PLCB1, PLCG2, PLCD4 and PLCE were not expressed. PLCB3, PLCB4, PLCD1, PLCD3, PLCH1 and PLCH2 were expressed. PLCB2 was not expressed in the samples of patients bearing the EWSR1-FLI1 translocation, and was expressed in the remaining samples. PLCG1 was not expressed in 20% of the samples (not overlapping).

SS samples: PLCB1, PLCG2, PLCD3 and PLCH1 were not expressed; was very slightly expressed. PLCB and PLCD1 were expressed. PLCB2, PLCG1 or PLCH2 were not expressed in 25% of the samples and expressed in the remaining samples (not overlapping). PLCB4 was expressed in 50% of the samples. PLCD4 was expressed in 25% of the samples.

Discussion

Research efforts were addressed to analyze the metabolism of bones and the signal transduction pathways involved in bone mineralization were actively studied (58-61), with special regard to the systems related to calcium metabolism (62).

Osteoblasts, MG-63 cells and SaOS-2 cells share similar PLC enzymes panel of expression, confirming those observations that have led to use the 2 osteosarcoma cell lines as osteoblasts experimental model. However, slight differences were detected in the expression of selected isoforms.

Confirming our previous reports (26-32), MG-63 expressed a number of PLC enzymes, including PLCB1, PLCB3, PLCB4, PLCG1, PLCG2, PLCD1, PLCD3, PLCE and inconstantly PLCB2. SaOS-2 expressed PLCB1, PLCB3, PLCB4, PLCG1, PLCG2, PLCD1, PLCD3, PLCE and PLCH1. Osteoblasts (26) expressed PLCB1, PLCB3, PLCB4, PLCG1, PLCG2, PLCD1, PLCD3, PLCD4 and PLCE, PLCH1 and PLCH2 were inconstantly expressed.

PLCD4 mRNA was differently detected. The mRNA transcript for PLCD4 was detected in osteoblasts, while it was absent in both MG-63 and SaOS-2 cells. The PLC δ4 protein was not detected in any of the analyzed cell lines.

Interestingly, in our previous reports PLC δ4 was inconstantly expressed in fibroblasts (13), was expressed in the Hs888 human metastatic cells and was absent in other human osteosarcoma cell lines. In previous reports, the transcription of PLCD4 was up-regulated after silencing of ezrin, a membrane protein interacting with actin which is involved in the metastatic spread of osteosarcoma (26-31). Moreover, PLCD4 absence in MG-63 cells did not change after administration of PLC inhibitors, indicating that it was constitutionally untranscribed (31).

PLC δ enzymes, the most primitive and evolutionary conserved, are known to be very sensitive to calcium and were described to play a key role in cell proliferation (9).In yeast and higher plants, PLC δ4 is involved in nutritional and environmental stresses (63,64).Moreover, recent observations suggested that PLC δ4 might be involved in the stress-induced response in endothelial cells (12,19-21). In the regenerating liver, PLC δ4 mRNA is expressed at higher levels than in normal resting liver, as well as in hepatoma cells, and in Src-transformed cells (9).PLC δ4 was demonstrated to regulate the liver regeneration in cooperation with nuclear protein kinase C (PKC) alpha and epsilon (65).

Moreover, PLC δ4 expression was abundant in a number of tumours, including astrocytomas (11,15,16). In breast cancer the abnormal expression of PLC δ4 contributes to carcinogenesis by up-regulating ErbB expression and activating the ERK pathway (66). Probably, the expression of PLC δ4 is a response to mitogenic signals or the isoform is expressed more abundantly in high-rate proliferating cells (67).In our present experiments, the transcript of PLCD4 was detected exclusively in osteoblasts, while it was absent in either the osteosarcoma cell lines. The presence of PLCD4 transcript in osteoblasts might confirm its role in metabolically active cells. However, the absence of PLCD4 expression in both MG-63 and SaOS-2 cells indicate that those cell lines cannot be perfectly compared to osteoblasts.

The presence of PLCD4 transcript in osteoblasts in the absence of the corresponding protein suggests that a complex regulation of PLC δ4 translation occurs in cells. Those aspects and the possible relationship with other PLC enzymes will require further investigations. In fact, up-regulation of PLCD4 was suggested to be related to the regulation of other PLC enzymes, probably PLC β1 (16-30).That might be due to the highest sensitivity of PLC δ isoforms to calcium concentration and suggest that an internal regulatory hierarchy might exist among PLC enzymes. That is an old issue, in fact for a long time it was hypothesized that PLC δ4 or some splice variants might act as negative regulators for PLC (68).

That represents an interesting point, in that also for the activity of PLC η isoforms calcium levels are critical (9,69,70). In the present experiments, osteoblasts differently expressed PLCH1 and PLCH2 compared to MG-63 and SaOS-2 cells.

PLC η1 acts as a signal amplifier in G protein-coupled receptor (GPCR)-mediated calcium signaling. Knocking down PLC η1, but not PLC-η2, significantly reduced the PLC pathway. PLC η1 is efficiently activated by intracellular calcium stores, suggesting that mobilization of calcium from the ER plays a critical role in PLC η1 activation (71). That might fit with our present observation that PLCD4 is expressed in osteoblasts. In fact, PLC δ4 is activated by low concentrations of calcium. Once activated, it can increase the intracellular calcium concentration via IP3 production, thus activating PLC η1 as a positive feed-back. However, despite the presence of PLCD4 transcript in osteoblasts, PLC δ4 protein was slightly detected within the cell performing immunofluorescence experiments.

Similarly within the cell, PLC η2 contributes to calcium dynamics (69) by transducing signals from mitochondria calcium. Alterations in the calcium levels modulate the activity of PLC η2, suggesting that this isoform may contributes to regulate the calcium signaling networking intracellular and extracellular stimuli. The sensitivity of PLC η2 to calcium might favour the amplification of intracellular calcium transients and/or crosstalk between storing compartments.

The subcellular distribution of the PLC enzymes also showed slight differences in the analysed cell lines. In particular, selected enzymes such as PLC β1 and either PLC η enzymes were differently stored within the cell. In osteoblasts, PLC β1 seems to be stored in vesicles partially resembling the endoplasmic reticulum distribution. In SaOS-2 cells, PLC η subfamily enzymes also seem to be stored in vesicles and the punctuate distribution of PLC η2 results less evident.

The present results confirm that osteoblasts, MG-63 cells and SaOS-2 cells share similar panel of expression of PLC enzymes, with notable exceptions indicating that MG-63 and SaOS-2 osteosarcoma cell lines might not represent an appropriate experimental model to analyse the signal transduction in osteoblasts.

Interesting differences were identified relative to the expression of PLCs in ES and SS. In fact, most ES and SS did not express PLCB1, which was expressed in most osteoblasts, MG-63 and SaOS-2 cells. Conversely, PLCB2, unexpressed in the cell lines, was expressed in some ES and SS. However, PLCH1 was expressed in SaOS-2 and inconstantly expressed in osteoblasts, while it was expressed in ES and unexpressed in SS.

The main genetic abnormalities described in ES and SS are actually considered as crucial elements for diagnosis (9,72,73). ES is defined by a balanced translocation that involves the EWSR1 gene (OMIM *133450; mapping on 22q12.2) and a member of the E-twenty-six (ETS) family of transcription factors, most frequently FLI1 (OMIM *193067; mapping in 11q24.3) or ERG (OMIM *165080; mapping in 21q22.2) (74,75). Beside the main translocations previously described, either tumour can bear additional secondary chromosomal abnormalities, such as gains of chromosomes and/or loss of chromosome regions (76-78).

Previous studies performed in epigenetically modified stable ES’ cells showed partial loss of endogenous EWSR1-FLI1 due to transfection with antisense EWSR1-FLI1 DNA plasmid (79). Interestingly, the expression of PLC β2 and PLC β3 resulted reduced in the transfected cells that showed EWSR1-FLI1 loss. PLCB3 maps on chromosome 11, region 11q13. However, the break point of the EWS-Fli-1 rearrangement usually involves the 11q24 region, probably distal to the PLCB3 locus. Conversely, PLCB2 gene maps in chromosome 15, in a region not commonly involved in the rearrangement. Those findings suggested that the impairment of the G-protein- mediated PI turnover could be responsible for the concomitantly observed suppression of transfected cells growth (79). That supported the hypothesis that signalling through Gq and PLC β isoforms might represent a crucial pathway in the transformation and growth of tumours (13-20,80).

In the present experiments, PLCB1 gene was not expressed in either ES or SS. Conversely it was not detected in cultured cells. PLC β1 was suggested to be mainly involved in inflammation (21-25,30), as well as in differentiation (81,82). Moreover, PLC β1 might be altered in breast cancer (83) and its partial or total lack might influence cancer progression in myeloid tissue (84). Previous studies (24-26) demonstrated that in osteosarcoma, PLCB1 increase is associated with the decrease of ezrin, which is thought to facilitate tumour progression, suggesting that PLC β1 might play an opposite role.

PLCB2 transcript was detected in 70% of SS samples and in 80% of ES samples. Remarkably, PLC β2 was not detected in ES samples bearing the EWSR1-ERG translocation. PLC β2 is mainly expressed in haematopoietic lineage cells (9).PLC β2 protein was also detected in normal skin fibroblasts (13), as well as in skin fibroblasts from patients presenting with hypertension (85). PLC β2 is thought to be a part of the IGF2-IGF2R/PLC β2 axis, which is involved in neovascularization (86). Previous reports also demonstrated a specific role of PLC β2 in osteosarcoma cells mechano-transduction and attachment, indicating that the presence of PLC β2 might be correlated with specific stimuli (85). The meaning of PLC β2 absence in ES carrying the EWSR1-ERG translocation needs further studies.

The most relevant difference observed in ES compared with SS regarded PLC ε and PLC η isoforms. PLCH1 and PLCH2 were expressed in ES samples. PLCH1 was absent and PLCH2 was present in 75% of the SS samples. Conversely, PLCE was not expressed in ES and was weakly expressed in SS. The role of PLC η subfamily is not fully highlighted. The alternate presence of one/both PLC η isoforms or PLC ε was described in other cell types, suggesting a sort of dualism. The role of PLC η isoforms needs further studies in order to be elucidated. In fact, different splicing isoforms were described, PLC η enzymes are considered very highly sensitive to calcium concentrations and probably interact with PLC isoforms belonging to other subfamilies in a complex manner.

Conclusion

Our results indicated that the expression of PLC enzymes differs in osteoblasts, ES and SS. Specific expression panel of PLCs suggests that selected isoforms might play a specific role in calcium metabolism in osteoblasts which differ from ES and SS. Rapid advances in molecular methodologies will allow to discover novel genetic events and help to refine diagnostic criteria, highlighting the biology of a number of tumours. The PI signal transduction pathway might deserve further research effort in order to investigate the role of PLC enzymes in the progression of sarcomas and also paving the way to novel diagnostic elements.

Authors’ Contribution

VRLV: experimental design, results elaboration, discussion, article writing. ML and ASdA: experiments. CDR: critical discussion.

Conflict of Interest Disclosures

All authors declare that there is no conflict of interests.

Funding/Support

None.

References

1. Hu F, Pan L, Zhang K, Xing F, Wang X, Lee I, et al. Elevation of extracellular Ca2+ induces store-operated calcium entry via calcium-sensing receptors: a pathway contributes to the proliferation of osteoblasts. PLoS One 2014;9(9):e107217. doi:10.1371/journal.pone.0107217. [Crossref]
2. Berridge MJ, Dupont G. Spatial and temporal signalling by calcium. Curr Opin Cell Biol 1994;6(2):267-74.
3. Divecha N, Irvine RF. Phospholipid signaling. Cell 1995;80(2):269-78.
4. Hisatsune C, Nakamura K, Kuroda Y, Nakamura T, Mikoshiba K. Amplification of Ca2+ signaling by diacylglycerol-mediatedinositol 1,4,5-trisphosphate production. J Biol Chem 2005;280(12):11723-30. doi:10.1074/jbc.M409535200. [Crossref]
5. Bunney TD, Katan M. PLC regulation: emerging pictures for molecular mechanisms. Trends Biochem Sci 2011;36(2):88-96. doi:10.1016/j.tibs.2010.08.003. [Crossref]
6. Fukami K, Inanobe S, Kanemaru K, Nakamura Y. Phospholipase C is a key enzyme regulating intracellular calcium and modulating the phosphoinositide balance. Prog Lipid Res 2010;49(4):429-37. doi:10.1016/j.plipres.2010.06.001. [Crossref]
7. Tang CH, Yang RS, Fu WM. Prostaglandin E2 stimulates fibronectin expression through EP1 receptor, phospholipase C, protein kinase C alpha, and c-Src pathway in primary cultured rat osteoblasts. J Biol Chem 2005;280(24):22907-16.
8. Rhee SG, Kim H, Suh PG, Choi WC. Multiple forms of phosphoinositide-specific phospholipase C and different modes of activation. Biochem Soc Trans 1991;19(2):337-41.
9. Suh PG, Park J, Manzoli L, Cocco L, Peak JC, Katan M, et al. Multiple roles of phosphoinositide-specific phospholipase C isozymes. BMB Rep 2008;41:415-434.
10. Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, et al. PLC z eta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development. Development 2002;129:3533-44.
11. Lo Vasco VR, Fabrizi C, Artico M, Cocco L, Billi AM, Fumagalli L, et al. Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes. J Cell Biochem 2007;100(4):952-959.
12. Lo Vasco VR, Pacini L, Di Raimo T, D’Arcangelo D, Businaro R. Expression of Phosphoinositide-specific phospholipase C isoforms in HUVEC. J Clin Pathol 2011;64(10):911-5. doi:10.1136/jclinpath-2011-200096. [Crossref]
13. Lo Vasco VR, Leopizzi M, Chiappetta C, Puggioni C, Di Cristofano C, Della Rocca C. Expression of Phosphoinositide-specific phospholipase C enzymes in human skin fibroblasts. Connect Tissue Res 2013;54(1):1-4. doi:10.3109/03008207.2012.712584. [Crossref]
14. Lo Vasco VR, Polonia P. Molecular cytogenetic interphase analysis of Phosphoinositide-specific Phospholipase C β1 gene in paraffin-embedded brain samples of major depression patients. J Affect Disord 2012;136(1-2):177-180.
15. Lo Vasco VR, Fabrizi C, Panetta B, Fumagalli L, Cocco L. Expression pattern and sub cellular distribution of phosphoinositide specific phospholipase C enzymes after treatment with U-73122 in rat astrocytoma cells. J Cell Biochem 2010;110(4):1005-12.
16. Lo Vasco VR, Fabrizi C, Fumagalli L, Cocco L. Expression of phosphoinositide specific phospholipase C isoenzymes in cultured astrocytes activated after stimulation with Lipopolysaccharide. J Cell Biochem 2010;109(5):1006-12. doi:10.1002/jcb.22480. [Crossref]
17. Lo Vasco VR, Leopizzi M, Chiappetta C, Businaro R, Polonia P, Della Rocca C, et al. Expression of phosphoinositide-specific phospholipase C enzymes in normal endometrium and in endometriosis. Fertil Steril 2012;98(2):410-4. doi:10.1016/j.fertnstert.2012.04.020. [Crossref]
18. Lo Vasco VR, Leopizzi M, Della Rocca C, Fais P, Montisci M, Cecchetto G. Impairment and reorganization of the phosphoinositide-specific phospholipase C enzymes in suicide brains. J Affect Disord 2015;174:324-8. doi:10.1016/j.jad.2014.12.006. [Crossref]
19. Lo Vasco VR, Leopizzi M, Puggioni C, Della Rocca C, I, Businaro R. Neuropeptide Y significantly reduces the expression of PLCB2, PLCD1 and moderately decreases selected PLC genes in endothelial cells. Mol Cell Biochem 2014;394(1-2):43-52. doi:10.1007/s11010-014-2079-2. [Crossref]
20. Lo Vasco VR, Leopizzi M, Puggioni C, Della Rocca C, Businaro R. Fibroblast growth factor acts upon the transcription of phospholipase C genes in human umbilical vein endothelial cells. Molecular and Cellular Biochemistry 2014;388(1):51-59. doi:10.1007/s11010-013-1898-x. [Crossref]
21. Lo Vasco VR, Leopizzi M, Chiappetta C, Puggioni C, Della Rocca C, Businaro R. Lypopolysaccharide down-regulates the expression of selected phospholipase C genes in cultured endothelial cells. Inflammation 2013;36(4):862-8. doi:10.1007/s10753-013-9613-3. [Crossref]
22. Lo Vasco VR, Longo L, Polonia P. Phosphoinositide-specific Phospholipase C ß1 gene deletion in bipolar disorder affected patient. J Cell Commun Signal 2013;7(1):25-9. doi:10.1007/s12079-012-0182-2. [Crossref]
23. Lo Vasco VR, Cardinale G, Polonia P. Deletion of PLCB1 gene in schizophrenia affected patients. J Cell Mol Med 2012;16(4):844-851.
24. Di Raimo T, Leopizzi M, Mangino G, Rocca CD, Businaro R, Longo L, et al. Different expression and subcellular localization of Phosphoinositide-specific Phospholipase C enzymes in differently polarized macrophages. J Cell Commun Signal 2016;10(4):283-93.
25. Lo Vasco VR, Leopizzi M, Di Maio V, Della Rocca C. U-73122 reduces the cell growth in cultured MG-63 ostesarcoma cell line involving phosphoinositide-specific phospholipases C. Springerplus 2016;5:156. doi:10.1186/s40064-016-1768-6. [Crossref]
26. Lo Vasco VR, Leopizzi M, Della Rocca C. Ezrin-related Phosphoinositide pathway modifies RhoA and Rac1 in human osteosarcoma cell lines. J Cell Commun Signal 2015;9(1):55-62.
27. Lo Vasco VR, Leopizzi M, Della Rocca C. Ezrin-related Phosphoinositide pathway modifies RhoA and Rac1 in human osteosarcoma cell lines. J Cell Commun Signal 2015;9(1):55-62. doi:10.1007/s12079-015-0265-y. [Crossref]
28. Lo Vasco VR, Leopizzi M, Puggioni C, Della Rocca C. Ezrin silencing remodulates the expression of Phosphoinositide-specific Phospholipase C enzymes in human osteosarcoma cell lines. J Cell Commun Signal 2014;8(3):219-29. doi:10.1007/s12079-014-0235-9. [Crossref]
29. Lo Vasco VR, Leopizzi M, Stoppoloni D, Della Rocca C. Silencing of phosphoinositide-specific phospholipase C ε remodulates the expression of the phosphoinositide signal transduction pathway in human osteosarcoma cell lines. Anticancer Res 2014;34(8):4069-75.
30. Lo Vasco VR, Leopizzi M, Chiappetta C, Puggioni C, Di Cristofano C, Della Rocca C. Expression of phosphoinositide-specific phospholipase C enzymes in human osteosarcoma cell lines. J Cell Commun Signal 2013;7(2):141-50.
31. Lo Vasco VR, Leopizzi M, Scotto D’Abusco A, Della Rocca C. Comparison of phosphoinositide-specific phospholipase C expression panels of human osteoblasts versus MG-63 and Saos Osteoblast-Like Cells. Avicenna Biomed J 2016;4(2):e34104.
32. Hofstaetter JG, Roschger A, Puchner SE, Dominkus M, Sulzbacher I, Windhager R, et al. Altered matrix mineralization in a case of a sclerosing osteosarcoma. Bone 2013;53(2):409-13. doi:10.1016/j.bone.2012.12.043. [Crossref]
33. Mackie EJ. Osteoblasts: novel roles in orchestration of skeletal architecture. Int J Biochem Cell Biol 2003;351301-5 .
34. Raggatt LJ, Partridge NC. Cellular and molecular mechanisms of bone remodeling. J Biol Chem 2010;285:25103-8 .
35. Matsuo K, Irie N. Osteoclast-osteoblast communication. Arch Biochem Biophys 2008;473:201-9 .
36. Bonewald LF, Schwartz Z, Swain LD, Boyan BD. Stimulation of matrix vesicle enzyme activity in osteoblast-like cells by 1,25(OH)2D3 and transforming growth factor beta (TGF beta). Bone Miner 1992;17(2):139-44.
37. Maillard C, Berruyer M, Serre CM, Dechavanne M, Delmas PD. Protein-S, a vitamin K-dependent protein, is a bone matrix component synthesized and secreted by osteoblasts. Endocrinology 1992;130(3):1599-604.
38. Schwartz Z, Dennis R, Bonewald L, Swain L, Gomez R, Boyan BD. Differential regulation of prostaglandin E2 synthesis and phospholipase A2 activity by 1,25-(OH)2D3 in three osteoblast-like cell lines (MC-3T3-E1, ROS 17/2.8, and MG-63). Bone 1992;13(1):51-8.
39. Fukayama S, Tashjian AH Jr. Direct modulation by estradiol of the response of human bone cells (SaOS-2) to human parathyroid hormone (PTH) and PTH-related protein. Endocrinology 1989;124(1):397-401.
40. Fukayama S, Bosma TJ, Goad DL, Voelkel EF, Tashjian AH Jr. Human parathyroid hormone (PTH)-related protein and human PTH: comparative biological activities on human bone cells and bone resorption. Endocrinology 1988;123(6):2841-8.
41. Rodan SB, Imai Y, Thiede MA, Wesolowski G, Thompson D, Bar-Shavit Z, et al. Characterization of a human osteosarcoma cell line (Saos-2) with osteoblastic properties. Cancer Res 1987;47(18):4961-6.
42. Esiashvili N, Goodman M, Marcus RB Jr. Changes in incidence and survival of Ewing sarcoma patients over the past 3 decades: surveillance epidemiology and end results data. J Pediatr Hematol Oncol 2008;30(6):425-30.
43. Mascarenhas L, Siegel S, Spector L, Arndt C, Femino D, Malogolowkin M. Malignant bone tumors. In: Bleyer A, O’Leary M, Barr R, eds. Cancer Epidemiology in Older Adolescents and Young Adults 15 to 29 Years of Age, Including SEER Incidence and Survival: 1975–2000. Bethesda, MD: National Cancer Institute; 2006:97–109.
44. Jawad MU, Cheung MC, Min ES, Schneiderbauer MM, Koniaris LG, Scully SP. Ewing sarcoma demonstrates racial disparities in incidence-related and sex-related differences in outcome: an analysis of 1631 cases from the SEER database, 1973–2005. Cancer 2009;115(15):3526-36.
45. Choi EY, Gardner JM, Lucas DR, McHugh JB, Patel RM. Ewing sarcoma. Semin Diagn Pathol 2014;31(1):39-47.
46. Parham DM, Hijazi Y, Steinberg SM, Meyer WH, Horowitz M, Tzen CY, et al. Neuroectodermal differentiation in Ewing’s sarcoma family of tumors does not predict tumor behavior. Hum Pathol 1999;30(8):911–918.
47. Folpe AL, Goldblum JR, Rubin BP, Shehata BM, Liu W, Dei Tos AP, et al. Morphologic and immunophenotypic diversity in Ewing family tumors: a study of 66 genetically confirmed cases. Am J Surg Pathol 2005;29(8):1025-33.
48. Nascimento AG, Unii KK, Pritchard DJ, Cooper KL, Dahlin DC. A clinicopathologic study of 20 cases of large-cell (atypical) Ewing0s sarcoma of bone. Am J Surg Pathol 1980;4(1):29-36.
49. Llombart-Bosch A, Machado I, Navarro S, Bertoni F, Bacchini P, Alberghini M, et al. Histological heterogeneity of Ewing0s sarcoma/PNET: an immunohistochemical analysis of 415 genetically confirmed cases with clinical support. Virchows Arch 2009;455(5):397–411. doi:10.1007/s00428-009-0842-7. [Crossref]
50. Machado I, Noguera R, Mateos EA, Calabuig-Fariñas S, López FI, Martínez A, et al. The many faces of atypical Ewing’s sarcoma. A true entity mimicking sarcomas, carcinomas and lymphomas. Virchows Arch 2011;458 (3):281–290. doi:10.1007/s00428-010-1023-4. [Crossref]
51. Kerouanton A, Jimenez I, Cellier C, Laurence V, Helfre S, Pannier S, et al. Synovial sarcoma in children and adolescents. J Pediatr Hematol Oncol 2014;36(4):257-62. doi:10.1097/MPH.0000000000000154. [Crossref]
52. Brennan B, Stevens M, Kelsey A, Stiller CA. Synovial sarcoma in childhood and adolescence: a retrospective series of 77 patients registered by the Children’s Cancer and Leukaemia Group between 1991 and 2006.Pediatr Blood Cancer 2010;55(1):85-90. doi:10.1002/pbc.22453. [Crossref]
53. Ferrari A, Gronchi A, Casanova M, Meazza C, Gandola L, Collini P, et al. Synovial sarcoma: a retrospective analysis of 271 patients of all ages treated at a single institution. Cancer 2004;101:627-34. doi:10.1002/cncr.20386. [Crossref]
54. Ferrari A, Casanova M. New concepts for the treatment of pediatric nonrhabdomyosarcoma soft tissue sarcomas. Expert Rev Anticancer Ther 2005;5:307-18.
55. Speth BM, Krieg AH, Kaelin A, Exner GU, Guillou L, von Hochstetter A, et al. Synovial sarcoma in patients under 20 years of age: a multicenter study with a minimum follow-up of 10 years. J Child Orthop 2011;5:335–342. doi:10.1007/s11832-011-0360-4. [Crossref]
56. Guillou L, Benhattar J, Bonichon F, Gallagher G, Terrier P, Stauffer E, et al. Histologic grade, but not SYT-SSX fusion type, is an important prognostic factor in patients with synovial sarcoma: a multicenter, retrospective analysis. J Clin Oncol 2004;22:4040–4050.
57. Orbach D, Mc Dowell H, Rey A, Bouvet N, Kelsey A, Stevens MC. Sparing strategy does not compromise prognosis in pediatric localized synovial sarcoma: experience of the International Society of Pediatric Oncology, Malignant Mesenchymal Tumors (SIOPMMT) Working Group. Pediatr Blood Cancer 2011;57(7):1130-6. doi:10.1002/pbc.23138. [Crossref]
58. Partridge NC, Bloch SR, Pearman AT. Signal transduction pathways mediating parathyroid hormone regulation of osteoblastic gene expression. J Cell Biochem 1994;55(3):321-7.
59. ten Dijke P, Fu J, Schaap P, Roelen BA. Signal transduction of bone morphogenetic proteins in osteoblast differentiation. J Bone Joint Surg Am 2003;85-A Suppl 3:34-8.
60. Yavropoulou MP, Yovos JG. The role of the Wnt signaling pathway in osteoblast commitment and differentiation. Hormones (Athens) 2007;6(4):279-94.
61. Lausson S, Cressent M. Signal transduction pathways mediating the effect of adrenomedullin on osteoblast survival. J Cell Biochem 2011;112(12):3807-15. doi:10.1002/jcb.23311. [Crossref]
62. Tatrai A, Lee SK, Stern PH. U-73122, a phospholipase C antagonist, inhibits effects of endothelin-1 and parathyroid hormone on signal transduction in UMR-106 osteoblastic cells. Biochim Biophys Acta 1994;1224(3):575-82.
63. Liu N, Fukami K, Yu H, Takenawa T. A new phospho- lipase Cδ4 is induced at S-phase of the cell cycle and appears in the nucleus. J Biol Chem 1996;271:355-60.
64. Fukami K, Takenaka K, Nagano K, Takenawa T. Growth factor-induced promoter activation of murine phospholipase Cδ4 gene. Eur J Biochem 2000;267:28–36.
65. Akutagawa A, Fukami K, Banno Y, Takenawa T, Kannagi R, Yokoyama Y, et al. Disruption of phospholipase Cdelta4 gene modulates the liver regeneration in cooperation with nuclear protein kinase C. J Biochem 2006;140(5):619-25. doi:10.1093/jb/mvj194. [Crossref]
66. Leung DW, Tompkins C, Brewer J, Ball A, Coon M, Morris V, et al. Phospholipase C delta-4 over- expression upregulates ErbB1/2 expression, Erk signaling path- way, and proliferation in MCF-7 cells. Mol Cancer 2004;3:15. doi:10.1186/1476-4598-3-15. [Crossref]
67. Ochocka AM, Pawelczyk T. Isozymes delta of phosphoinositide-specific phospholipase C and their role in signal transduction in the cell. Acta Biochim Pol 2003;50(4):1097-110.
68. Nagano K, Fukami K, Minagawa T, Watanabe Y, Ozaki C, Takenawa T. A novel phospholipase C delta4 (PLCdelta4) splice variant as a negative regulator of PLC. J Biol Chem 1999;274(5):2872-9.
69. Popovics P, Beswick W, Guild SB, Cramb G, Morgan K, Millar RP, et al. Phospholipase C-η2 is activated by elevated intracellular Ca2+ levels. Cell Signal 2011;23(11):1777-84. doi:10.1016/j.cellsig.2011.06.012. [Crossref]
70. Popovics P, Lu J, Nadia Kamil L, Morgan K, Millar RP, Schmid R, et al. A canonical EF-loop directs Ca(2+) -sensitivity in phospholipase C-η2. J Cell Biochem 2014;115(3):557-65. doi:10.1002/jcb.24690. [Crossref]
71. Kim JK, Choi JW, Lim S, Kwon O, Seo JK, Ryu SH, et al. Phospholipase C-η1 is activated by intracellular Ca2+ mobilization and enhances GPCRs/PLC/Ca2+ signaling. Cell Signal 2011;23(6):1022-9. doi:10.1016/j.cellsig.2011.01.017. [Crossref]
72. Höglund M, Gisselsson D, Mandahl N, Mitelman F. Ewing tumours and synovial sarcomas have critical features of karyotype evolution in common with epithelial tumours. Int J Cancer 2005;116(3):401-6.
73. Höglund M, Frigyesi A, Säll T, Gisselsson D, Mitelman F. Statistical behavior of complex cancer karyotypes. Genes Chromosomes Cancer 2005;42(4):327-41. doi:10.1002/gcc.20143. [Crossref]
74. Delattre O, Zucman J, Plougastel B, Desmaze C, Melot T, Peter M, et al. Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature 1992;359(6391):162–165. doi:10.1038/359162a0. [Crossref]
75. de Alava E, Lessnick SL, Sorensen PH. Ewing Sarcoma. In: Fletcher CDM, Bridge JA, Hogendoorn PC, eds. WHO Classification of Tumours of Soft Tissue and Bone. Lyon: IARC Press; 2013:306-9.
76. Ohno T, Rao VN, Reddy ES. EWS/Fli-1 chimeric protein is a transcriptional activator. Cancer Res 1993;53(24):5859-63.
77. Ohno T, Ouchida M, Lee L, Gatalica Z, Rao VN, Reddy ES. The EWS gene, involved in Ewing family of tumors, malignant melanoma of soft parts and desmoplastic small round cell tumors, codes for an RNA binding protein with novel regulatory domains. Oncogene 1994;9(10):3087-97.
78. Armengol G, Tarkkanen M, Virolainen M. Recurrent gains of 1q, 8 and 12 in the Ewing family of tumours by comparative genomic hybridization. Br J Cancer 1997;75(10):1403-9.
79. Dohjima T, Ohno T, Banno Y, Nozawa Y, Wen-yi Y, Shimizu K. Preferential down-regulation of phospholipase C-beta in Ewing’s sarcoma cells transfected with antisense EWS-Fli-1. Br J Cancer 2000;82(1):16-9.
80. Beekman A, Helfrich B, Bunn PA Jr, Heasley LE. Expression of catalytically inactive phospholipase Cbeta disrupts phospholipase Cbeta and mitogen-activated protein kinase signaling and inhibits small cell lung cancer growth. Cancer Res 1998;58(5):910-3.
81. Faenza I, Bavelloni A, Fiume R, Santi P, Martelli AM, Billi AM, et al. Expression of Phospholipase C β Family Isoenzymes in C2C12 myoblasts during terminal differentiation. J Cell Physiol 2004;200(2):291-6. doi:10.1002/jcp.20001. [Crossref]
82. O’Carroll SJ, Mitchell MD, Faenza I, Cocco L, Gilmour RS. Nuclear PLCbeta1 is required for 3T3-L1 adipocyte differentiation and regulates expression of the cyclin D3-cdk4 complex. Cell Signal 2009;21(6):926-35.
83. Molinari C, Medri L, Follo MY, Piazzi M, Mariani GA, Calistri D, et al. PI-PLCβ1 gene copy number alterations in breast cancer. Oncol Rep 2012;27(2):403-8. doi:10.3892/or.2011.1529. [Crossref]
84. Kaminskas E, Farrell A, Abraham S, Baird A, Hsieh LS, Lee SL, et al. Approval summary: azacitidine for treatment of myelodysplastic syndrome subtypes. Clin Cancer Res 2005;11(10):3604-8.
85. Hoberg M, Gratz HH, Noll M, Jones DB. Mechanosensitivity of human osteosarcoma cells and phospholipase C beta2 expression. Biochem Biophys Res Commun 2005;333(1):142-9.
86. Maeng YS, Choi HJ, Kwon JY, Park YW, Choi KS, Min JK, et al. Endothelial progenitor cell homing: prominent role of the IGF2-IGF2R-PLCbeta2 axis. Blood 2009;113(1):233-43. doi:10.1182/blood-2008-06-162891. [Crossref]