OPN Gene Research Articles

Overexpression of hsa-miR-125b during osteoblastic differentiation does not influence levels of Runx2, osteopontin, and ALPL gene expression.

Multipotent mesenchymal stromal cells (MSCs) were first isolated from bone marrow and then from various adult tissues including placenta, cord blood, deciduous teeth, and amniotic fluid. MSCs are defined or characterized by their ability to adhere to plastic, to express specific surface antigens, and to differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Although the molecular mechanisms that control MSC proliferation and differentiation are not well understood, the involvement of microRNAs has been reported. In the present study, we investigated the role of miR-125b during osteoblastic differentiation in humans. We found that miR-125b increased during osteoblastic differentiation, as well as Runx2 and ALPL genes. To study whether the gain or loss of miR-125b function influenced osteoblastic differentiation, we transfected MSCs with pre-miR-125b or anti-miR-125b and cultured the transfected cells in an osteoblastic differentiation medium. After transfection, no change was observed in osteoblastic differentiation, and Runx2, OPN, and ALPL gene expression were not changed. These results suggest that the gain or loss of miR-125b function does not influence levels of Runx2, OPN, and ALPL during osteoblastic differentiation.

Alterations in Osteopontin Modify Muscle Size in Females in Both Humans and Mice

An osteopontin (OPN; SPP1) gene promoter polymorphism modifies disease severity in Duchenne muscular dystrophy, and we hypothesized that it might also modify muscle phenotypes in healthy volunteers. Gene association studies were carried out for OPN (rs28357094) in the FAMuSS cohort (n = 752; mean ± SD age = 23.7 ± 5.7 yr). The phenotypes studied included muscle size (MRI), strength, and response to supervised resistance training. We also studied 147 young adults that had carried out a bout of eccentric elbow exercise (age = 24.0 ± 5.2 yr). Phenotypes analyzed included strength, soreness, and serum muscle enzymes. In the FAMuSS cohort, the G allele was associated with 17% increase in baseline upper arm muscle volume only in women (F = 26.32; P = 5.32 × 10), explaining 5% of population variance. In the eccentric damage cohort, weak associations of the G allele were seen in women with both baseline myoglobin and elevated creatine kinase. The sexually dimorphic effects of OPN on muscle were also seen in OPN-null mice. Five of seven muscle groups examined showed smaller size in OPN-null female mice, whereas two were smaller in male mice. The query of OPN gene transcription after experimental muscle damage in mice showed rapid induction within 12 h (100-fold increase from baseline), followed by sustained high-level expression through 16 d of regeneration before falling to back to baseline. OPN is a sexually dimorphic modifier of muscle size in normal humans and mice and responds to muscle damage. The OPN gene is known to be estrogen responsive, and this may explain the female-specific genotype effects in adult volunteers.

Abstract 3879: Osteopontin (SPP1/OPN) alternative splice variants and metastatic potential in esophageal adenocarcinoma.

Abstract Objective: The incidence of esophageal adenocarcinoma (EAC) has increased over 300% in the past decades in western countries. EAC is characterized by an early metastasis, with a 5-year survival rate of only 19%, making the identification of clinically-targetable proteins critical for improving patient outcome. Osteopontin (SPP1/OPN) is a secreted glycosylated phosphoprotein that is found overexpressed in many cancer types including EAC. OPN protein functions in cell adhesion and cell migration by regulating cell-matrix interactions and cellular signaling through binding with integrin and CD44 receptors. However, little is known about the role of OPN in EAC invasion and metastasis. Methods: We analyzed the OPN gene expression in a series of 46 esophageal samples including esophageal Barrett's, low-grade dysplasia, high-grade dysplasia and adenocarcinomas mRNA using Affymetrix U133A arrays. Real-time RT-PCR was performed to validate array results and examine a larger cohort of 130 EAC samples. Tissue microarrays (TMA) were made containing 64 EACs, eight lymph node metastases, eight dysplastic and 11 none dysplastic Barrett's mucosa sections from 59 patients. Primers flanking the potential gene alternative splice regions were designed and the in-frame transcript variants were sequenced. Expression of OPN in EAC cell lines was analyzed using RT-PCR and western blot analysis. Ectopic OPN expression of splice variants and OPN knockdown in EAC cell lines have been performed to examine its roles in tumor invasion and EMT assays. Results: OPN is highly overexpressed in EAC and overexpression of the gene is associated with poorer survival in patients. Three OPN splice variants were identified with their expression either proportionally increased or differentially expressed in the primary EAC and EAC-derived cells. Highly overexpressed OPN is a transcriptional event as no OPN gene amplification was found in the corresponding EACs. In addition, abundant OPN protein was found primarily in tumor cells in the tissue sections using immunohistochemical (IHC)-TMA assays. OPN variants differ in their N-terminal thrombin fragment with either exon 6 or 5 being absent in OPN-b or OPN-c, respectively. We found that EAC cells treated with the medium containing different OPN variants demonstrated distinctive morphological phenotypes. We further identified that OPN variants played different roles in tumor cell proliferation, epithelial-mesenchymal transition (EMT), and chemosensitivity. Conclusion: Upregulation of OPN is predominant event in primary EACs and associated with poorer survival in patients. The alternative splice variants demonstrate differential tumor invasion, EMT transition and chemosensitivity in EAC cells. Citation Format: Zhuwen Wang, Kimmy Leverenz, Dafydd G. Thomas, Amy L. Myers, Andrew C. Chang, Mark B. Orringer, Thomas J. Giordano, Jules Lin, David G. Beer, Lin Lin. Osteopontin (SPP1/OPN) alternative splice variants and metastatic potential in esophageal adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3879. doi:10.1158/1538-7445.AM2013-3879

Single nucleotide polymorphism of promoters of candidate genes controlling porcine productivity indices

Associations of single-nucleotide polymorphisms (SNPs) in promoters of specialized genes of lipid metabolism (Scd, Lep), myogenesis (Myod, Myf-6), and board-spectrum protein regulator gene (Opn) with porcine meat-fat productivity indices are analyzed. The revealed associations depend on the origin of the animals (are different in two- and three-breed crosses) and their sex. The greatest number of associations is observed between SNPs of the Opn gene promoter and backfat thickness and meat productivity indices. The data obtained indicate that the investigated SNP genotypes can be used for increasing the effectiveness of predicting the productive potential of animals with consideration of their origin and sex.

Abstract 89: Osteopontin-c and osteopontin-b splicing isoforms activate prostate cancer progression features

Abstract Osteopontin (OPN) is one of the proteins overexpressed in prostate cancer (PCa) and is involved in tumorigenesis and metastasis. Alternative splicing of the OPN gene generates three protein splicing isoforms (OPN-SI), designated as OPNa, OPNb and OPNc, which have demonstrated specific roles in different tumor models. However, the roles of each of these isoforms have not been previously characterized in PCa. Our previous study reported that OPN-SI were overexpressed in PCa tissues, strongly associated with PCa occurrence and with tumor cell differentiation. Besides, OPNc was the most upregulated variant. We have also demonstrated that OPNc splicing isoform can contribute for PCa diagnosis and prognosis. Based on these results, the present study aimed to evaluate the putative roles of OPN-SI in PCa biology using in vitro and in vivo functional assays. PC-3 was stably transfected with expression vectors containing OPNa, OPNb and OPNc, as well as empty vector control. PC-3 cells overexpressing each construct were analyzed for in vivo tumor growth and in relation to different aspects mimicking tumor progression, such as cell proliferation, migration, invasion and soft agar colony formation. OPNc and OPNb overexpressing cells significantly activated enhanced xenograft tumor growth and PC-3 proliferation, migration, invasion, and soft agar colony formation, as well as the expression of several cancer related genes. Additionally, we have also shown that some of the OPNc and OPNb-dependent pro-tumorigenic roles are mediated by PI3K/Akt signaling pathway. Inhibition of this pathway by using LY294002 specifically inhibited tumor progression features evoked by OPNc and OPNb overexpression. According to our data, OPNc and OPNb roles on favoring different aspects of PCa progression suggest that these isoforms contribute to the physiopathology of prostate cancer progression and tumorigenesis. Altogether, the data open possibilities of new therapeutic approaches for prostate cancer that selectively downregulate OPNc and OPNb, altering its properties favoring prostate tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 89. doi:1538-7445.AM2012-89

Osteopontin Biomarker in Inflammatory Bowel Disease, Animal Models and Target for Drug Discovery

Inflammatory bowel disease (IBD) is an immune-mediated disease caused by abnormal mucosal T-cell response to bacteria in the intestinal lumen. In Crohn’s disease (CD), mucosal T cells exhibit the TH1 phenotype, whereas ulcerative colitis (UC) is believed to have a TH2 phenotype [1]. In the absence of a single test, calculating disease activity in IBD remains a complicated task. An accurate and reliable marker for measuring CD or UC activity for routine clinical practice and in clinical trials has to be based on clinical judgment of histologic and endoscopic imaging. Therefore, validated accurate and objective measures of inflammatory activity in IBD using a reliable, non-invasive biomarker is needed [2]. Proteonome biomarkers are used in the diagnostics of IBD, but they are expensive and not all of them are validated [3]. Several recent reports have suggested an important role for osteopontin (OPN) in the pathogenesis of IBD as well as its possible use as a biomarker [4–10]. Masuda et al. demonstrated that expression of OPN is significantly elevated in the mucosa of patients with UC [6]. Elevated OPN expression in UC was demonstrated using immunohistochemistry and ultrastructure analysis [7, 8]. In CD, Gassler et al. reported an elevated OPN expression in plasma cells, but not in epithelial cells [9]. Moreover, Sato et al. observed an elevated plasma OPN and significant correlation with disease activity in CD, but not in UC [10]. Osteopontin, a Th1 cytokine, is up-regulated in relation to the severity of the disease [11]. Moreover, since it is an adhesive glycoprotein containing the peptide sequence glycine ± arginine ± aspartate ± serine it promotes cell attachment [12]. Osteopontin and tumor necrosis factor receptor signaling (TNFRS) F14 are genes associated with fibrosis/cell adhesion molecules [1, 13]. Blockade of these cell adhesion molecule interactions prevents the trafficking of leukocytes across the vascular endothelium and, subsequently, into the parenchymal tissue [14–16]. Alpha-4 integrins bind additional ligands in tissues, including OPN and epitopes of fibronectin. This is the rationale for using the mechanism of monoclonal antibodies in clinical practice. The scope may be to suppress ongoing inflammatory reactions in diseased tissues by inhibition of a4-positive leukocytes with ligands. Inhibiting the existing inflammatory activity at the disease site, along with inhibition of further recruitment of immune cells into inflamed tissue, via interaction with adhesion molecules such as the vascular adhesion molecule will reduce the inflammation and intestinal fibrosis [17–19]. On the basis of these findings, several researchers described animal models that can mimic the OPN-induced colitis in vivo as well as block OPN-induced inflammation [20–23]. In this issue is the article ‘‘Osteopontin ablation attenuates progression of TNBS-induced colitis model.’’ In this study, Oz et al. elegantly demonstrate that by ablating the OPN activity the colitis is suppressed [24]. The developmental stages of colonic inflammation in a modified trinitrobenzene sulfonic acid (TNBS) model that mimics Crohn’s disease have been defined as acute, subacute, and chronic. The researchers used OPN deficient mice, with a targeted disruption of the opn gene in a C57BL/6. TNBStreated wildtype mice developed severe colitis but OPNdeficient mice were significantly protected. M. G. Neuman (&) In Vitro Drug Safety and Biotechnology, Department of Pharmacology and Toxicology, Faculty of Medicine, Institute of Drug Research, University of Toronto, MaRS Discovery District, 101 College Street, Suite 300, Lab 351, South Tower, Toronto, ON M5G 1L7, Canada e-mail: m_neuman@rogers.com

Basic fibroblast growth factor lentiviral vector-transfected sheep bone marrow mesenchymal stem cells and non-specific osteogenic gene expression.

In the present study, sheep bone marrow stem cells (BMSCs) were transfected with basic fibroblast growth factor (bFGF)-lentivirus in order to investigate the influence of bFGF on osteogenic gene expression. bFGF‑transfected BMSCs (experimental group) and non-transfected BMSCs (control group) were also transfected with a green fluorescent protein gene in order to measure the transfection efficiency of the (bFGF)-lentivirus using flow cytometry. Using quantitative polymerase chain reaction assays, the changes in expression from three genes (osteopontin, OPN; osteocalcin, OC; and collagen-I) in BMSCs from the experimental and control groups were measured. Transfection efficiency was 87.3% in the experimental group and 1.1% in the control group. OPN gene expression was high in BMSCs from the experimental group. However, there was no significant difference in OPN expression between BMSCs from the control and experimental group (P>0.05). Collagen-I expression was significantly lower in the experimental group compared with that in the control groups (P<0.05). By contrast, OC expression was significantly higher in BMSCs from the experimental group compared with that in the control group (P<0.05). Changes in osteogenic gene expression indicated that the BMSCs from the experimental group had better osteogenic ability, as compared with the control cells. Therefore, bFGF-transfected cells may be useful seed cells for bone tissue engineering.

The impact of osteopontin gene variations on multiple sclerosis development and progression.

Osteopontin is a proinflammatory molecule, modulating TH1 and TH17 responses. Several reports suggest its involvement in multiple sclerosis (MS) pathogenesis. We previously reported that OPN gene variations at the 3′ end are a predisposing factor for MS development and evolution. In this paper, we extended our analysis to a gene variation at the 5′ end on the −156G > GG single nucleotide polymorphism (SNP) and replicated our previous findings at the 3′ end on the +1239A > C SNP. We found that only +1239A > C SNP displayed a statistically significant association with MS development, but both +1239A > C and −156G > GG had an influence on MS progression, since patients homozygous for both +1239A and −156GG alleles displayed slower progression of disability and slower switch to secondary progression than those carrying +1239C and/or −156G and those homozygous for +1239A only. Moreover, patients homozygous for +1239A also displayed a significantly lower relapse rate than those carrying +1239C, which is in line with the established role of OPN in MS relapses.

Repair of rabbit ulna segmental bone defect using freshly isolated adipose-derived stromal vascular fraction

Stromal vascular fractions (SVF) from adipose tissue have heterogeneous cell populations, and include multipotent adipose-derived stem cells. The advantages of using of SVF include the avoidance of an additional culture period, a reduced risk of extensive cell contamination, and cost-effectiveness. Unilateral 20-mm mid-diaphyseal segmental defects in rabbit ulna were treated with one of the following: polylactic glycolic acid (PLGA) scaffold alone (group 1, control), a PLGA scaffold with undifferentiated SVF cells (group 2), or a PLGA scaffold with osteogenically differentiated SVF cells (group 3). At 8 weeks after implantation, five rabbits in each treatment group were killed to assess bone defect healing by plain radiography, quantitative microcomputed tomography and histology. The SVF cells were well grown on PLGA scaffolds and expressed type I collagen and alkaline phosphatase (ALP). The intensity of ALP and OPN gene expressions in osteogenic medium culture were increased from 14 days to 28 days. In vivo evaluations at 8 weeks showed that treatment of SVF cells with or without osteogenic differentiation resulted in more bone formation in the critically sized segmental defects than PLGA scaffold alone. Osteogenically differentiated SVF cells significantly enhanced bone healing compared with undifferentiated SVF cells. Adipose-derived stromal SVF showed osteogenic potential in vitro. Accordingly, SVF could provide a cell source for bone tissue engineering. However, treatment with uncultured SVF cells on bone healing was not satisfactory in the in vivo animal model.

Abnormal Expression of the ERG Transcription Factor in Prostate Cancer Cells Activates Osteopontin

Osteopontin (OPN) is an extracellular matrix glycophosphoprotein that plays a key role in the metastasis of a wide variety of cancers. The high level of OPN expression in prostate cells is associated with malignancy and reduced survival of the patient. Recent studies on prostate cancer (PCa) tissue have revealed recurrent genomic rearrangements involving the fusion of the 5' untranslated region of a prostate-specific androgen-responsive gene with a gene coding for transcription factors from the ETS family. The most frequently identified fusion gene is TMPRSS2:ERG, which causes ERG protein overexpression in PCa cells. ERG is a transcription factor linked to skeletogenesis. This study was designed to test whether ERG and the product of the TMPRSS2:ERG fusion gene modulate OPN gene expression in PCa cells. To characterize ERG and TMPRSS2:ERG transcriptional activity of OPN, we focused on ETS binding sites (EBS) localized in conserved regions of the promoter. Using in vitro and in vivo molecular assays, we showed that ERG increases OPN expression and binds to an EBS (nt -115 to -118) in the OPN promoter. Moreover, stable transfection of prostate tumor cell lines by TMPRSS2:ERG upregulates endogenous OPN expression. Finally, in human prostate tumor samples, detection of the TMPRSS2:ERG fusion gene was significantly associated with OPN overexpression. Taken together, these data suggest that OPN is an ERG-target gene in PCa where the abnormal expression of the transcription factor ERG, due to the TMPRSS2:ERG fusion, disturbs the expression of genes that play an important role in PCa cells and associated metastases.

NF-κB– and AP-1–Mediated DNA Looping Regulates Osteopontin Transcription in Endotoxin-Stimulated Murine Macrophages

Osteopontin (OPN) is expressed by various immune cells and modulates both innate and adaptive immune responses. However, the molecular mechanisms that control opn gene expression, especially at the chromatin level, remain largely unknown. We have previously demonstrated many specific cis- and trans-regulatory elements that determine the extent of endotoxin (LPS)-mediated induction of OPN synthesis in murine macrophages. In the present study, we confirm that NF-κB also plays an important role in the setting of LPS-stimulated OPN expression through binding to a distal regulatory element. Importantly, we demonstrate that LPS stimulates chromosomal loops in the OPN promoter between NF-κB binding site and AP-1 binding site using chromosome conformation capture technology. The crucial role of NF-κB and AP-1 in LPS-stimulated DNA looping was confirmed, as small interfering RNA knock-down of NF-κB p65 and AP-1 c-Jun exhibited decreased levels of DNA looping. Furthermore, we demonstrate that p300 can form a complex with NF-κB and AP-1 and is involved in DNA looping and LPS-induced OPN expression. Therefore, we have identified an essential mechanism to remodel the local chromatin structures and spatial conformations to regulate LPS-induced OPN expression.

Determination of the differentiation capacities of murines' primary mononucleated cells and MC3T3-E1 cells

BackgroundThe main morphological features of primitive cells, such as stem and progenitor cells, are that these cells consists only one nucleus. The main purpose of this study was to determine the differentiation capacities of stem and progenitor cells. This study was performed using mononucleated cells originated from murine peripheral blood and MC3T3-E1 cells. Three approaches were used to determine their differentiation capacities: 1) Biochemical assays, 2) Gene expression analysis, and 3) Morphological observations.ResultsWe found that both cells were able to differentiate into mature osteoblasts, as assayed by ALP activity. RT-PCR analysis showed the activation of the Opn gene after osteoblast differentiation. Morphological observations of both cells revealed the formation of black or dark-brown nodules after von Kossa staining. Nevertheless, only mononucleated cells showed the significant increase in TRAP activity characteristic of mature osteoclasts. The osteoclast-specific CatK gene was only upregulated in mononucleated cells. Morphological observations indicated the existence of multinucleated osteoclasts. Sca-1 was activated only in undifferentiated mononucleated cells, indicating that the cells were hematopoietic stem cells. In both cell lines, the housekeeping Gapdh gene was activated before and after differentiation.ConclusionThe isolated mononucleated cells were able to differentiate into both osteoblasts and osteoclasts; indicating that they are stem cells. On the other hand, MC3T3-E1 cells can only differentiate into osteoblasts; a characteristic of progenitor cells.

Differential Expression of Intracellular and Secreted Osteopontin Isoforms by Murine Macrophages in Response to Toll-like Receptor Agonists

Osteopontin (OPN), expressed by various immune cells, modulates both innate and adaptive immune responses. Different immune cells have shown differential expression of the two isoforms of OPN: secreted form of OPN (sOPN) and intracellular form of OPN (iOPN). However, the molecular mechanisms that control opn gene expression and the OPN isoforms produced by immune cells remain largely unknown. In this study, we demonstrate that OPN mRNA and protein expression are significantly up-regulated upon stimulation with TLR agonists in macrophages. Interestingly, we find that macrophages constitutively express the secreted form of OPN (sOPN), while the intracellular form of OPN (iOPN) is induced following the stimulation with TLR agonists. Phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and c-Jun NH(2)-terminal kinase (JNK) that are activated by LPS stimulation were shown to upregulate OPN expression. In addition, chromatin immunoprecipitation (CHIP) assays showed that AP-1 binds to the proximal AP-1 site in the OPN promoter from LPS-stimulated macrophages. Mutation of the AP-1 site in OPN promoter completely ablates LPS-induced OPN promoter activation. Knockdown of c-Jun and c-Fos expression by small interfering RNA (siRNA) significantly decreases LPS-induced OPN expression. Stable cell lines with iOPN overexpression and knockdown showed that TLR-induced iOPN expression is a negative regulator for interferon-beta (IFN-beta) production. Our findings provide new insight into the transcriptional regulation of opn gene and further clarify the isoforms and functions of OPN produced by macrophages.

Abstract 234: Transcription factor LSF is a novel oncogene for hepatocellular carcinoma (HCC)

Abstract While the incidence of overall cancers is decreasing, Hepatocellular Carcinoma (HCC) is one of the few cancers the incidence of which is on an increase. The mortality rate of HCC parallels that of incidence since HCC is a highly aggressive cancer with extremely limited currently available therapeutic strategies. Understanding the molecular mechanism of hepatocarcinogenesis will help develop novel, targeted and more effective therapeutic regimen to counteract this fatal disease. Here, we demonstrate that the cellular transcriptional factor Late SV40 Factor (LSF) functions as an oncogene in HCC. The level of LSF expression is significantly increased in human HCC patients compared to matched non-neoplastic controls and normal liver and LSF expression significantly correlates with the stages and grades of HCC. Ectopic overexpression of LSF in nontumorigenic human HCC cells leads to highly aggressive, multi-organ metastatic tumors while inhibition of LSF in aggressive human HCC cells profoundly inhibits tumorigenic and metastatic properties in athymic nude mice. Affymetrix microarray studies identified novel LSF-downstream genes that are associated with cell growth, angiogenesis, invasion, metastasis, chemoresistance, and senescence. Osteopontin, a key molecule mediating tumor progression, especially metastasis, was identified as a downstream target gene of LSF. Promoter-reporter and chromatin immunoprecipitation analyses confirmed that LSF transcriptionally upregulates OPN by binding to its promoter. Knockdown of OPN gene in LSF-overexpressing HCC cells markedly inhibits LSF-mediated tumor progression including metastasis. This is the first report on the novel function of LSF as an oncogene in any cancer indication. Since LSF expression correlates with the advanced stages of the disease, it might be employed as a diagnostic/prognostic marker for HCC. The DNA binding domain of LSF might be targeted with small molecule inhibitors that might be developed as potential HCC therapeutic. Our findings thus have significant translational implications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 234.

Differentiation of Bone Marrow Stromal Cells into Osteoblasts in a Self-assembling Peptide Hydrogel: In Vitro and In Vivo Studies

A prerequisite of tissue engineering approaches with regard to autograft is a suitable scaffold that can harbor cells and signals. Conventionally, such scaffolds have been prepared as 3D scaffolds prefabricated from synthetic or natural biomaterials. RAD16 has been introduced as a new biomaterial, where synthetic peptides self-assemble to form a hydrogel. In this study, RAD16 was examined in terms of osteogenic efficacy and feasibility of ectopic mineralization. Two hundred and seventy-one RAD16 was cocultured with 1 × 10(6) bone marrow cells from the femurs of 6-week-old Wistar male rats in alpha minimum essential medium supplemented with or without dexamethasone. Second, the same volume of the RAD16 construct hosting the cells with or without hydroxyapatite (HA) particles was treated in the dexamethasone medium as well, prepared in a Teflon tube, and implanted subcutaneously. Cell proliferation was prominent in the RAD16 coculture with dexamethasone at 1 week and significantly decreased by 2 weeks, whereas the other combinations remained or inclined, and their osteogenic differentiation was accelerated up to 2 weeks, as seen in increasing alkaline phosphatase (ALP) activity and mRNAs of ALP, OPN, and OCN. The RAD16 implant prepared with HA particles allowed more osteoblast-like cells and blood cells to grow inside, which was accompanied by elevating OPN gene expression and the stronger peak of VEGF gene expression at 2 weeks. Furthermore, more OPN mRNA signal was detected around the RAD16 containing HA particles by 4 weeks. On the other hand, the RAD16 alone represented lower expression of OPN gene. During the experiment, however, no ectopic mineralization was observed in both groups. Conclusively, it was suggested that the RAD16 showed feasibility of serving as a matrix for osteogenic differentiation of cocultured bone marrow cells in vitro and in vivo. Proceeding of exploration and modification of RAD16 are continuously required for cell-based tissue engineering.

Differentiation analyses of adult suspension mononucleated peripheral blood cells of Mus musculus

BackgroundThe purpose of this study is to determine whether isolated suspension mouse peripheral mononucleated blood cells have the potential to differentiate into two distinct types of cells, i.e., osteoblasts and osteoclasts.ResultsDifferentiation into osteoblast cells was concomitant with the activation of the Opn gene, increment of alkaline phosphatase (ALP) activity and the existence of bone nodules, whereas osteoclast cells activated the Catk gene, increment of tartrate resistant acid phosphatase (TRAP) activity and showed resorption activities via resorption pits. Morphology analyses showed the morphology of osteoblast and osteoclast cells after von Kossa and May-Grunwald-Giemsa staining respectively.ConclusionsIn conclusion, suspension mononucleated cells have the potentiality to differentiate into mature osteoblasts and osteoclasts, and hence can be categorized as multipotent stem cells.

The Osteopontin Gene +1239A/C Single Nucleotide Polymorphism is Associated with Type 1 Diabetes Mellitus in the Italian Population

Secreted phosphoprotein 1, also known as Osteopontin (Opn), is a proinflammatory cytokine involved in the TH1 response and is highly expressed in the islets and pancreatic lymph nodes of non-obese diabetic mice before the onset of diabetes. In humans, typing of the +1239A/C single nucleotide polymorphism (SNP) in the 3UTR of the Opn gene (SPP1) showed that +1239C carriers displayed higher Opn serum levels than +1239A homozygotes and a higher risk of developing autoimmune/lymphoproliferative syndrome, multiple sclerosis, and systemic lupus erythematosus. The aim of this work is to evaluate whether +1239A/C is also associated with type 1 diabetes mellitus (T1DM). We typed +1239A/C in an initial cohort of 184 T1DM patients and 361 controls, and confirmed our data in a second cohort of 513 patients and 857 controls. In both cohorts, +1239C carriers displayed a significantly higher risk of T1DM than +1239A homozygotes (combined cohorts: OR=1.63, 95 percent CI: 1.34-1.97). Clinical analysis did not detect any differences between patients carrying or not +1239C in terms of gender distribution and age at T1DM diagnosis. These data suggest that SPP1 variants marked by +1239C are associated with T1DM development in the Italian population. The predisposing effect may depend on its effect on Opn levels.

Expression of &lt;I&gt;OPN&lt;/I&gt; gene during different lactation stages in mammary gland of dairy goat and its effect on growth of MCF-7 cell line

To investigate the expression pattern and preliminary function of OPN gene in mammary gland of dairy goat during different lactation stages, using b-actin gene as the internal control, the SYBR Green quantitative real-time PCR (QPCR) analysis was conducted to determine the mRNA expression of OPN gene in mammary gland at the 28th, 60th, 100th, 190th, 270th and 330th day after kidding. Recombinant plasmid of pcDNA3.1-OPN was constructed by inserting the fragment of OPN gene into eukaryotic expression vector pcDNA3.1 and used to transfect the MCF-7 cell line following the restrictive endonuclease cleavage and sequence identification of the target gene segment, the effect of OPN gene on MCF-7 cell proliferation was assessed by MTT analysis. The results indicated that OPN gene exhibited the higher expression level in early (28 d) and late (190 d) lactation stages and the lowest level at dry stage (330 d), which demonstrated a high-low-high-low pattern. There was a significant difference (P < 0. 05) in the proliferation between OPN gene transfected and non-transfected MCF-7 cells, which suggested that the expression of OPN gene could stimulate the proliferation of MCF-7 cells.

Egr-1 upregulates OPN through direct binding to its promoter and OPN upregulates Egr-1 via the ERK pathway

Early growth response factor-1 (Egr-1) and osteopontin (OPN) play important roles in the migration and proliferation of vascular smooth muscle cells (VSMC), but little is known about their relationship. Therefore, we transfected VSMCs with either Egr-1 cDNA, Opn cDNA, a DNA enzyme designed to target Egr-1 (ED5), or antisense Opn oligodeoxynucleotides and examined changes in Egr-1 and OPN expression at the mRNA and protein levels. OPN expression levels were increased in cells that were stably transfected with Egr-1 cDNA. By contrast, both Egr-1 and OPN expression were reduced when ED5 was transfected into Egr-1-expressing cells. Similarly, Opn transfection upregulated Egr-1 levels, while Opn anti-sense oligodeoxynucleotide transfection decreased Egr-1 expression. ChIP analysis showed that Egr-1 binds to the Opn gene promoter. Furthermore, treatment with the extracellular-regulated protein kinase (ERK) inhibitor PD98059 inhibited the upregulation of Egr-1 by OPN. We find that Egr-1 and OPN positively regulate each other in VSMCs.

507. TGFβ REGULATES ENDOMETRIOSIS-LIKE LESION DEVELOPMENT IN MICE INDEPENDENTLY OF THE RUNX-2/OPN PATHWAY

TGFβ is likely to significantly influence endometriotic lesion development, as TGFβ KO/SCID mice with no host-derived TGFβ activity have smaller human ectopic endometrial lesions than control mice. TGFβ potentially acts via RUNX2, a transcription factor that directly upregulates OPN transcription in osteoblasts, as we have identified RUNX-2 and OPN gene expression at high levels in nude mouse endometriosis-like lesions, in human endometrial stomal cell cultures and in the stroma of endometriotic tissues. We hypothesised that inhibition of RUNX-2 would suppress OPN production and result in reduction of endometriotic lesion formation and size. As P38/MAPK inhibitors suppress TGF-β mediated RUNX-2 transcription, we utilised the nude mouse model to test whether the P38/MAPK inhibitor FR167653 would suppress OPN production in endometriotic tissues resulting in smaller lesions.FR167653 (30 mg/kg twice a day) or placebo was administered for either 10 or 14 days to nude mice (16 in each group) implanted with human endometrial tissue xenografts from 4 different women. The size and weight of the lesions were measured and immunohistochemical analysis of OPN, αSMA (myofibroblasts) and F4/80 (macrophages) was carried out. There was no difference in size or weight of the lesions, and there was no overt difference in any of the staining parameters explored. The inhibition of p38/MAPK did not alter the size of the nude mouse lesions nor OPN staining within these lesions despite being administered at a maximal therapeutic dose. This suggests that TGFβ regulation of endometriotic lesion development is mediated by an alternate molecular pathway.