Genetic variations in the SPP1 promoter affect gene expression and the level of osteopontin secretion into bovine milk

Osteopontin (OPN) is now recognized as an important cytokine and extracellular integrin‐binding protein at the crossroads of inflammation and homeostasis. In a previous study, we found that OPN gene (SPP1) polymorphisms are associated with milk performance traits and somatic cell score (SCS), a parameter used to estimate the genetic value of udder health in dairy cattle. In this study, we assessed whether the genetic variations had an impact on SPP1 promoter activity, immune response and the level of OPN secreted into milk. The influence of DNA polymorphisms on the promoter activity of SPP1 was confirmed in vitro. To measure the impact of the genetic variations on OPN secretion into milk, we measured OPN levels in both plasma and milk throughout lactation. Cows were grouped by the OPN haplotypes associated with a high (H2 × H3) or low (H1 × H4) SCS. For both H2 × H3 and H1 × H4, the OPN level in plasma remained low throughout lactation, although the concentration in the milk of H1 × H4 cows increased more in late lactation. Moreover, the macrophages of H1 × H4 cows expressed a lower SPP1 and proinflammatory IL6 in response to infection. Regarding the immune cell response, cows with the genetic potential to secrete higher OPN levels during late lactation had macrophages expressing fewer proinflammatory cytokines, a situation that might explain the genetic association with low somatic cells. Although OPN's favorable roles during late lactation remain to be elucidated, the tissue remodeling properties associated with OPN may be beneficial for reducing the incidence of infection during the transition period in lactating cows.     

人膝骨关节炎软骨下骨OPN的表达及其意义

目的：通过检测人膝骨关节炎裸露软骨下骨中OPN的表达,探讨OPN在OA发病及病情进展中的意义。方法：选取接受膝关节置换手术的膝关节骨关节炎患者软骨下骨标本50例,采用综合评分法对OA患者进行严重程度分级,分为轻、中、重度三组,取正常膝关节软骨下骨（股骨髁关节面）10例作为正常软骨下骨对照;对标本进行免疫组织化学染色,用SPSS17．0统计软件包分析各组间OPN表达的差异及OA患者OPN表达与综合评分、K-L分期的相关性。结果：人膝骨关节炎裸露软骨下骨OPN表达明显高于正常软骨下骨组,差异有统计学意义（P〈0．01）;OPN在轻、中、重度膝骨关节炎裸露软骨下骨的表达差异有统计学意义（P〈0．05）;膝骨关节炎裸露软骨下骨OPN的表达与骨关节炎的综合评分、K—L分期呈正相关。结论：OA患者膝关节软骨下骨OPN表达与疾病严重程度呈正相关,提示OPN在骨关节炎发病及病情进展中可能起作用。     

结直肠癌肝转移患者血清OPN、YKL-40、HGF和VEGF-A水平及其危险因素分析

目的:检测结直肠癌肝转移患者血清骨桥蛋白(Osteopontin,OPN)、人类软骨糖蛋白-39(Human cartilage gp-39,YKL-40)、肝细胞生长因子(Hepatocyte growth factor,HGF)和血管内皮生长因子A(Vascular endothelial growth factor A,VEGF-A)水平,并分析直肠癌患者发生肝转移的危险因素。方法:收集56例发生肝转移的直结肠癌患者和50例未发生肝转移的直结肠癌患者,采用酶联免疫法检测两组患者血清OPN、YKL-40、HGF和VEGF-A的水平。收集所有患者的临床资料,分析直肠癌患者发生肝转移的危险因素。结果:结直肠癌肝转移患者血清OPN、YKL-40、HGF和VEGF-A水平均显著高于未发生肝转移的直结肠癌患者(P0.05);糖尿病、肿瘤最大直径、病理类型、分化程度、浸润深度、淋巴结转移、盆腹膜种植、肝外转移、吸烟史均与结直肠癌发生肝转移相关(P0.05);而HGF、VEGF-A、糖尿病、肿瘤最大直径≥5 cm、淋巴结转移、盆腹膜种植、肝外转移是直肠癌发生肝转移的独立危险因素(P0.05)。结论:结直肠癌肝转移患者血清OPN、YKL-40、HGF和VEGF-A明显升高,HGF、VEGF-A、糖尿病、肿瘤最大直径≥5 cm、淋巴结转移、盆腹膜种植、肝外转移是直肠癌发生肝转移的独立危险因素。     

The concordance test emerges as a powerful tool for identifying quantitative trait nucleotides: lessons from BTA6 milk yield QTL

The lack of conventions for confirming the discovery of quantitative trait nucleotides in livestock was evidenced by the proposals of mutations in two different genes ( SPP1 and ABCG2 ) as the underlying functional mutation for a major quantitative trait locus (QTL) for milk concentration on bovine chromosome 6 (BTA6). Of these conflicting candidates, SPP1 was excluded by follow-up studies and by the data described here. A simple test for concordance of the zygosity state between QTL segregation status and the candidate polymorphism was shown, in this case, to be a critical step towards establishing the proof. If a given sample effectively represents the genetic variation across the QTL region, haplotype-based concordance may further enhance the functionality and resolution power of this test, allowing identification of the causative gene.     

Human amelogenin up-regulates osteogenic gene expression in human bone marrow stroma cells

Extracts of enamel matrix proteins are used to regenerate periodontal tissues. Amelogenin, the most abundant enamel protein, plays an important role in the regeneration of these tissues. However, the molecular mechanisms by which amelogenin contributes to periodontal regeneration remain unknown. Using primary human bone marrow stroma cells (hBMSCs) transduced with lentivirus encoding human amelogenin (hAm), we performed genome-wide expression profiling to analyze the effects of hAm transduction on the regulation of genes involved in osteogenic differentiation. Our results revealed that BMP-2, BMP-6, OPN and VEGFC were up-regulated. These results suggest that hAm may be a key element in regulating hBMSCs osteogenic differentiation.     

Pre-treatment of cattle semen or oocytes with purified milk osteopontin affects in vitro fertilization and embryo development

This study was designed to investigate the effects of pre-incubating cattle spermatozoa or matured oocytes with purified osteopontin (OPN) from cattle milk on fertilization in cattle and embryonic development in vitro. There were two different experiments, semen from six mature Holstein bulls (Bos Taurus) was frozen with different concentrations of OPN (0, 1, 10, 100 μg/mL). Matured cattle oocytes were also pre-treated with OPN (0, 10, 100 μg/mL). In both experiments, pre-treated oocytes or frozen semen, was processed for in vitro fertilization and embryo development. Significantly more oocytes were fertilized when using frozen semen with 10 μg/mL OPN (bull 2 = 85 ± 4% and bull 5 = 78 ± 4%) than without OPN (bull 2 = 75 ± 4% and bull 5 = 69 ± 4%). Those bulls also had increase in cleavage and embryo development (bull 2 = 85 ± 3%, 41 ± 1.9%; bull 5 = 76 ± 2%, 37 ± 1.8%) compared with control (bull 2 = 75 ± 3%, 30 ± 2%; bull 5 = 68 ± 2%, 29 ± 2%). Incubating matured oocytes in 10 μg/mL OPN (87 ± 3%) and 100 μg/mL OPN (88 ± 3%) significantly increased fertilization than control (73 ± 3%). OPN also improve cleavage, and embryo development in treatments with 10 μg/mL OPN (82.7 ± 1.3%; 31.7 ± 1.4%) and 100 μg/mL OPN (85.8 ± 1.3%; 33.8 ± 1.5%) when compared with control (74.1 ± 1.3%; 24.2 ± 1.2%). These data suggest that both, spermatozoa from some bulls and oocytes may associate with OPN, suggesting a facilitory role on in vitro fertilization and embryo development.     

Suppression of host Th1-type granulomatous inflammation by Taenia solium metacestodes is related to down-regulation of osteopontin gene expression

Inflammation and granuloma formation in human neurocysticercosis has been attributed to Th1-type immune responses of the host. In the present murine model, over 94% of Taenia solium metacestodes were viable and elicited no granulomatous inflammation, whereas parasites killed by praziquantel treatment elicited rapid granuloma formation that calcified within 2weeks. Osteopontin (OPN) is a Th1-related cytokine that is up-stream of IL-12 and which may play an essential role in granuloma formation and calcification. OPN mRNA expression was down-regulated in tissues surrounding viable cysticerci, but was up-regulated in inflammatory tissues surrounding degenerating cysticerci. Moreover, co-culture with a viable cysticercus or ES products from these metacestodes led to a decrease in OPN, IFN-gamma and IL-12 expression, whereas co-culture with somatic proteins enhanced OPN expression by leukocytes. Addition of recombinant mouse OPN (rmOPN) counteracted the down-regulation of IL-12 and IFN-gamma mRNA expression, but not OPN mRNA expression, in leukocyte cultures. Furthermore, injection of rmOPN into the tissues surrounding implanted cysticerci enhanced inflammatory responses while a similar injection of an anti-rmOPN antibody reduced inflammation. These findings suggest that the suppression of host Th1-type granulomatous inflammation by ES products from T. solium metacestodes is related to down-regulation of OPN gene expression.     

Positive regulation of the Egr-1/osteopontin positive feedback loop in rat vascular smooth muscle cells by TGF-β, ERK, JNK, and p38 MAPK signaling

Previous studies identified a positive feedback loop in rat vascular smooth muscle cells (VSMCs) in which early growth response factor-1 (Egr-1) binds to the osteopontin (OPN) promoter and upregulates OPN expression, and OPN upregulates Egr-1 expression via the extracellular signal-regulated protein kinase (ERK) signaling pathway. The current study examined whether transforming growth factor-β (TGF-β) activity contributes to Egr-1 binding to the OPN promoter, and whether other signaling pathways act downstream of OPN to regulate Egr-1 expression. ChIP assays using an anti-Egr-1 antibody showed that amplification of the OPN promoter sequence decreased in TGF-β DNA enzyme-transfected VSMCs relative to control VSMCs. Treatment of VSMCs with PD98059 (ERK inhibitor), SP600125 (JNK inhibitor), or SB203580 (p38 MAPK inhibitor) significantly inhibited OPN-induced Egr-1 expression, and PD98059 treatment was associated with the most significant decrease in Egr-1 expression. OPN-stimulated VSMC cell migration was inhibited by SP600125 or SB203580, but not by PD98059. Furthermore, MTT assays showed that OPN-mediated cell proliferation was inhibited by PD98059, but not by SP600125 or SB203580. Taken together, the results of the current study show that Egr-1 binding to the OPN promoter is positively regulated by TGF-β, and that the p38 MAPK, JNK, and ERK pathways are involved in OPN-mediated Egr-1 upregulation.