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Inflammatory bowel disease (IBD) is a formidable disease due to its complex pathogenesis. Macrophages, as a major immune cell population in IBD, are crucial for gut homeostasis. However, it is still unveiled how macrophages modulate IBD. Here, we found that LIM domain only 7 (LMO7) was downregulated in pro-inflammatory macrophages, and that LMO7 directly degraded 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) through K48-mediated ubiquitination in macrophages. As an enzyme that regulates glycolysis, PFKFB3 degradation led to the glycolytic process inhibition in macrophages, which in turn inhibited macrophage activation and ultimately attenuated murine colitis. Moreover, we demonstrated that PFKFB3 was required for histone demethylase Jumonji domain-containing protein 3 (JMJD3) expression, thereby inhibiting the protein level of trimethylation of histone H3 on lysine 27 (H3K27me3). Overall, our results indicated the LMO7/PFKFB3/JMJD3 axis is essential for modulating macrophage function and IBD pathogenesis. Targeting LMO7 or macrophage metabolism could potentially be an effective strategy for treating inflammatory diseases.
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BACKGROUND: The research on neovascularization in diabetic retinopathy is mostly limited to vascular endothelial growth factor, but 6-phosphofructoklnase-2/fructose-2,6-diphosphatase (PFKFB3) also plays a certain role. OBJECTIVE: To Investigate the effect of PFKFB3-SÍRNA on human umbilical vein endothelial cells (HUVECs) In high glucose environment. METHODS: HUVECs were divided Into four groups: Normal glucose control group (5.5 mmol/L glucose), normal glucose+PFKFB3-slRNA group (5.5 mmol/L glucose+PFKFB3-siRNA), high glucose group (30 mmol/L glucose), high glucose+PFKFB3-slRNA group (30mmol/L glucose+PFKFB3-slRNA). Western blot assay was used to detect the silencing effect of PFKFB3 expression. PFKFB3 with optimal silencing effect was selected for subsequent experiments. The tubule formation was detected by in vitro tubule formation assay. The expression of PFKFB3 mRNA was detected by real-time fluorescent quantitative PCR. The expression of PFKFB3 and AKT protein was detected by western blot. RESULTS AND CONCLUSION: PFKFB3-SÍRNA significantly inhibited the expression of PFKFB3 (P 0.05). Compared with the high glucose group, the tube formation ability of the high glucose+PFKFB3-siRNA group was significantly increased (P 0.05). Compared with the high glucose group, the ratio of p-AKT/AKT protein in the high glucose+PFKFB3-siRNA group was increased (P < 0.01). To conclude, siRNA silencing of PFKFB3 gene expression can inhibit the expression of PFKFB3 and improve tube formation in HUVECs. The mechanism may be related to the down-regulation of AKT expression.
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Objective To investigate the expression of 6-phosphofructo-2-kinase /fructose-2,6-bisphosphatase-3 (PFKFB3) and sphingosine 1-phosphate receptor 2 (S1 P2) in the retina of rats with type 2 diabetes mellitus (DM) and to explore the correlation of tertiary butylhydroquinone (tBHQ) with PFKFB3 and S1P2.Methods Together 60 male SD rats were divided into normal control group (NC group),diabetes mellitus group (DM group) and tBHQ group.Type 2 DM model was induced in the latter two groups.The rats in tBHQ group were given 10 g · L-1 tBHQ in high-fat and highsugar diet 1 week after successful modeling,while DM rats were fed with high-fat and high-sugar diet continuously.At 4 weeks and 12 weeks after tBHQ intervention,blood samples were taken from the hearts of rats in each group,and serum contents of fasting plasma glucose (FPG) and fasting serum insulin (FINs) were measured.Immunohistochemistry and qRT-PCR were taken to detect the distribution and expression of PFKFB3,S1 P2 and vascular endothehal growth factor (VEGF) mRNA and protein in the retina,and TUNEL methods were used to detect apoptosis index of retinal ganglion cells of rats in each group.Results There were significant difference in the FPG and FINs levels of the three groups at 4 weeks and 12 weeks (all P =0.000).Light microscopy test showed that positive expressions of PFKFB3,S1 P2 and VEGF protein were found in all groups at 4 and 12 weeks,which were mainly located in the retinal ganglion cell layer and the inner nuclear layer.Immunohistochemistry and qRT-PCR showed that the difference in relative expressions of PFKFB3,S1P2,VEGF protein and mRNA in the retina at different times after modeling were statistically significant (all P ＜ 0.05).At 4 and 12 weeks,the expression levels of PFKFB3,S1P2 and VEGF in DM group were higher than those in NC group,but their expressions in tBHQ group were significantly downregulated when compared with DM group,and all differences were statistically significant (all P ＜ 0.05).Compared with 4 weeks,PFKFB3,S1 P2 and VEGF mRNA and protein in DM group were overexpressed at 12 weeks,and the expression level of S1 P2 in tBHQ group at 12 weeks was lower than that at 4 weeks,and the differences were statistically significant (all P ＜ 0.05).TUNEL assays showed that there was significant difference in the apoptotic index (AI) of retinal ganglion cells of rats in each group at different times after modeling (all P ＜ 0.05).DM group had higher AI than NC group (P ＜ 0.05),and tBHQ group was lower than DM group (P ＜ 0.05);Compared with 4 weeks,DM group had increased AI at 12 weeks while tBHQ group had decreased AI (both P ＜ 0.05).Conclusion PFKFB3,S1P2 and VEGF are involved in the pathological process of the retina of type 2 DM rats,which may play a role through the PFKFB3 / VEGF / S1P2 signaling pathway and may be related to the apoptosis of retinal ganglion cells.And it is indicated that tBHQ has a protective effect on the retina of type 2 DM rats.
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Objective To evaluate the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) in adenoid cystic carcinoma and the effects of PFKFB3 on the proliferation,migration,invasion and tumorigenesis in vivo of adenoid cystic carcinoma cells.Methods Immunohistochemistry and Westem blotting were applied to detect the expression of PFKFB3 in 29 cases of adenoid cystic carcinoma tissues and 10 cases of para-carcinoma normal salivary gland tissues.PFKFB3 in adenoid cystic carcinoma ACC-2 cells was silenced by adenovirus vector.The effects of PFKFB3 silence on cell proliferation,migration,invasion and tumorigenesis in vivo and distant metastasis of adenoid cystic carcinoma cells were evaluated by cell proliferation assay,wound healing assay,Transwell assay,xenografted mice model and lung metastasis mice model.Results The results of immunohistochemical staining showed that the positive expression rate of PFKFB3 in adenoid cystic carcinoma tissues was 93.1％ (27/29),and the positive expression rate of PFKFB3 in normal salivary gland tissues was 20.0％ (2/10),with a significant difference (x2 =20.84,P ＜ 0.001).The results of methyl thiasolyl tetrazolium (MTT) assay showed that,compared with ACC-2 group,the proliferation activity of cancer cells with silence of PFKFB3 (PFKFB3--ACC-2) was significantly suppressed for 72 h (1.8 ± 0.2 vs.4.7 ± 0.8,t =3.582,P =0.001).The results of wound healing assay showed that after scraping cells away for 24 h,the number of cells in the scratch area was 99.8 ± 13.2 in the PFKFB3--ACC-2 group,which was significantly less than that in the ACC-2 group (263.0 ± 97.4,t =2.868,P =0.029).Transwell results indicated that the number of cells passing through matrigel was 17.6 ± 2.1 in the PFKFB3--ACC-2 group,which was less than that in the ACC-2 group (28.6 ± 3.8,t =4.452,P =0.004).The tumor volume in the PFKFB3--ACC-2 [(623.5 ± 134.1) mm3] was smaller than that in the ACC-2 group [(1 621.0 ±278.1) mm3,t =4.213,P =0.001].More pulmonary metastases were found in the ACC-2 group thanPFKFB3--ACC-2group(18.1±3.2vs.4.1±2.2,t=6.322,P=0.001).Condusion The expression of PFKFB3 is higher in adenoid cystic carcinoma tissue than normal salivary gland tissue,and the highly expressed PFKFB3 plays a driving role in the proliferation,migration,invasion and metastasis in adenoid cystic carcinoma.
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@#[Abstract] Objective: Toevaluatetheexpressionof6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3(PFKFB3) in malignant glioma tissues and the effects of inhibitor of PFKFB3(PFK15) on the proliferation, migration, invasion, clone formation and tumorigenesis of H4 cells. Methods: Malignant brain glioma tissues and corresponding paratumor tissues from 31 patients, who were hospitalized in Department of Neurosurgery,Ankang Hospital of Traditional Chinese Medicine during February 1, 2015 to January 31, 2016 for operative treatment, were collected for this study. Immunohistochemistry and western blotting assays were applied to detect the expression of PFKFB3 in collected tissues. PFKFB3 in H4 cells were blocked by PFK15 (1.25, 2.5, 5.0 μmol/L). The effect of PFK15 on proliferation, migration, clone formation and tumorigenesis of H4 cells were determined by MTT assay, EdU incorporation assay, wound healing assay, Transwell assay, colone formation assay and in vivo xenograft bearing nude mice model respectively. Results: Positive expression rate of PFKFB3 was significantly higher in malignant glioma tissues compared with normal adjacent tissues[(80.60±8.98)% vs (41.57±10.16)%, P<0.05]. The results of MTT assay and EdU incorporation assay indicated that PEK15 significantly inhibited the proliferation of H4 cells in a concentration dependent manner. The migration, invasion and clone formation activity of H4 cells were significantly reduced by treatment with PFK15 (all P<0.05). In tumor bearing nude mice, the tumor volume of mice treated with PFK15 was significantly smaller than that of mice from control group ([254.15±154.25] vs [801.52±224.25] mm3, P<0.05). Conclusion: PFKFB3 was highly expressed in malignant glioma tissues. Blocking of PFKFB3 by PFK15 significantly reduced the malignant biological behaviors and tumorigenesis of H4 cells in vitro and in vivo, which may serve as a promising target for the treatment of malignant gliomas.
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Angiogenesis is essential for the occurrence, growth and metastasis of tumors. The differentiation and neovascularization of vascular endothelial cells depend on the energy supply by metabolism and its regulation, especially the glycolysis pathway is very important for vascular sprouting which is a starting point in the process of tumor angiogenesis. It has been found that phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3), a key rate-limiting enzyme of glycolysis pathway, has strong kinase activity. If the activity of PFKFB3 is inhibited, the rate of glycolysis can be reduced, thereby the vascular sprouting in tumor angiogenesis can be blocked. It has been demonstrated that PFKFB3 inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) can prominently reduce glycolysis and inhibit the pathologic angiogenesis. Therefore, in recent years, it becomes a new research hotspot in the field of cancer therapy to inhibit or destroy the metabolism of tumor vascular endothelial cells for the purpose of curing cancer. This review summarizes the effect of PFKFB3 in glycolysis of endothelial cells on tumor angiogenesis, and the research progress in PFKFB3 as a target for cancer treatment.