Wnt/β-catenin signaling is critical for various cellular processes in multiple cell types,including osteoblast(OB)differentiation and function.Exactly how Wnt/β-catenin signaling is regulated in OBs remain elusive.A...Wnt/β-catenin signaling is critical for various cellular processes in multiple cell types,including osteoblast(OB)differentiation and function.Exactly how Wnt/β-catenin signaling is regulated in OBs remain elusive.ATP6AP2,an accessory subunit of V-ATPase,plays important roles in multiple cell types/organs and multiple signaling pathways.However,little is known whether and how ATP6AP2 in OBs regulates Wnt/β-catenin signaling and bone formation.Here we provide evidence for ATP6AP2 in the OB-lineage cells to promote OB-mediated bone formation and bone homeostasis selectively in the trabecular bone regions.Conditionally knocking out(CKO)ATP6AP2 in the OB-lineage cells(Atp6ap2^(Ocn-Cre))reduced trabecular,but not cortical,bone formation and bone mass.Proteomic and cellular biochemical studies revealed that LRP6 and N-cadherin were reduced in ATP6AP2-KO BMSCs and OBs,but not osteocytes.Additional in vitro and in vivo studies revealed impairedβ-catenin signaling in ATP6AP2-KO BMSCs and OBs,but not osteocytes,under both basal and Wnt stimulated conditions,although LRP5 was decreased in ATP6AP2-KO osteocytes,but not BMSCs.Further cell biological studies uncovered that osteoblastic ATP6AP2 is not required for Wnt3a suppression ofβ-catenin phosphorylation,but necessary for LRP6/β-catenin and N-cadherin/β-catenin protein complex distribution at the cell membrane,thus preventing their degradation.Expression of activeβ-catenin diminished the OB differentiation deficit in ATP6AP2-KO BMSCs.Taken together,these results support the view for ATP6AP2 as a critical regulator of both LRP6 and N-cadherin protein trafficking and stability,and thus regulatingβ-catenin levels,demonstrating an un-recognized function of osteoblastic ATP6AP2 in promoting Wnt/LRP6/β-catenin signaling and trabecular bone formation.展开更多
Background: LDL receptor-related protein-1(LRP1) is a cell-surface receptor that functions in diverse physiological pathways. We previously demonstrated that hepatocyte-specific LRP1 deficiency(hLRP1KO) promotes diet-...Background: LDL receptor-related protein-1(LRP1) is a cell-surface receptor that functions in diverse physiological pathways. We previously demonstrated that hepatocyte-specific LRP1 deficiency(hLRP1KO) promotes diet-induced insulin resistance and increases hepatic gluconeogenesis in mice. However, it remains unclear whether LRP1 regulates hepatic glycogenesis.Methods: Insulin signaling, glycogenic gene expression, and glycogen content were assessed in mice and HepG2 cells. The pcDNA 3.1 plasmid and adeno-associated virus serotype 8 vector(AAV8) were used to overexpress the truncated β-chain(βΔ) of LRP1 both in vitro and in vivo.Results: On a normal chow diet, hLRP1KO mice exhibited impaired insulin signaling and decreased glycogen content. Moreover, LRP1 expression in HepG2 cells was significantly repressed by palmitate in a dose-and time-dependent manner. Both LRP1 knockdown and palmitate treatment led to reduced phosphorylation of Akt and GSK3β, increased levels of phosphorylated glycogen synthase(GYS), and diminished glycogen synthesis in insulin-stimulated HepG2 cells, which was restored by exogenous expression of the βΔ-chain. By contrast, AAV8-mediated hepatic βΔ-chain overexpression significantly improved the insulin signaling pathway, thus activating glycogenesis and enhancing glycogen storage in the livers of high-fat diet(HFD)-fed mice.Conclusion: Our data revealed that LRP1, especially its β-chain, facilitates hepatic glycogenesis by improving the insulin signaling pathway, suggesting a new therapeutic strategy for hepatic insulin resistance-related diseases.展开更多
目的:探讨低密度脂蛋白受体相关蛋白1(low density lipoprotein receptor-related protein 1,LRP1)在肺癌细胞中的表达水平及其生物学意义。方法:常规培养人类肺癌A549细胞,RT-PCR法检测A549细胞中LRP1的mRNA水平。构建针对LRP1的siRNA...目的:探讨低密度脂蛋白受体相关蛋白1(low density lipoprotein receptor-related protein 1,LRP1)在肺癌细胞中的表达水平及其生物学意义。方法:常规培养人类肺癌A549细胞,RT-PCR法检测A549细胞中LRP1的mRNA水平。构建针对LRP1的siRNA感染细胞,利用绿色荧光标记载体,通过荧光显微镜观察感染效率,利用RT-PCR检测敲减效率,Western blot检测LRP1表达水平变化。选用A549/LRP1 siRNA细胞系进行功能实验,利用CCK-8实验检测细胞增殖能力的变化,划痕实验检测细胞迁移能力的变化,Transwell侵袭小室实验检测细胞侵袭能力的变化。结果:从定量PCR结果可以看出,A549细胞中,相对于NC组,KD组LRP1基因敲减效率为77.7%,KD组细胞中LRP1的mRNA水平明显低于NC组,差异具有统计学意义(P<0.05);Western blot检测结果显示,KD组LRP1表达水平相对于NC组显著下调。CCK-8检测结果表明相对于NC组,KD组于Day 5的细胞增殖倍数显著下降,差异具有统计学意义(P<0.05)。划痕实验结果显示,相比NC组,KD组细胞划痕8小时、24小时迁移率明显降低,差异具有统计学意义(P<0.05)。Transwell侵袭实验结果显示,在侵袭小室内孵育24 h后KD组细胞侵袭转移率明显降低,差异具有统计学意义(P<0.05)。结论:LRP1对于肺癌A549细胞的生物学功能起着重要作用。下调LRP1能够抑制A549细胞的增殖、迁移及侵袭能力。对于LRP1分子上下游信号通路研究,在肺癌的诊断与治疗领域有一定的临床参考价值。展开更多
Objective: To study the multidrug resistance (MDR) mechanism of lung resistance protein (LRP) gene in hepatocellular carcinoma (HCC), and the relations among the expression of the LRP gene and clinicopathologic featur...Objective: To study the multidrug resistance (MDR) mechanism of lung resistance protein (LRP) gene in hepatocellular carcinoma (HCC), and the relations among the expression of the LRP gene and clinicopathologic features, the influence of α-fetoprotein (AFP), and prognosis of patients who received adjuvant chemotherapy after resection of HCC. Methods: The expression of the LRP gene encoding LRP and mRNA LRP was detected in tissues from 54 untreated patients with HCC, adjacent tissues from 24 patients with HCC and archival paraffin-embedded tissues from 12 patients with posthepatitic cirrhosis. The relationship between the LRP gene expression and the change of AFP level was analyzed in the 24 postoperative HCC patients whose AFP was measured after 2 weeks. All of the HCC patients were followed up. Results: The percentage of positive expression of LRP and mRNA LRP in the 3 tissues was 61.1%, 33.3%, 16.7%, and 75.9%, 37.5%, 33.3% respectively. There was significant difference between the untreated HCC tissue and other tissues (P<0.05). No difference existed between the LRP gene expression and clinicopathologic findings, age, sex, and tumor size (P>0.05), but the expression was related to the degree of differentiation of HCC (P<0.05). The effective rate of AFP in the LRP gene positive expression group or in postoperative chemotherapeutic patients was very lower than that in the negative group (P<0.05). Although the mean survival time of postoperative HCC patients in negative LRP gene expression group was longer than that of positive group, there was no difference between them (P<0.05). Conclusion: LRP gene expression is related to MDR of HCC and initiates the intrinsic MDR. Detection of LRP gene expression is of great guiding significance in accessing chemotherapeutic resistance of HCC. As an index to chemotherapy of HCC, detection of LRP expression provides evidence for making individual chemotherapeutic treatment,and reversing MDR in HCC. Although LRP gene expression correlates with the tumor differential degree (P<0.05), it perhaps does not relate with the prognosis of HCC patients.展开更多
基金supported in part by grants from the National Institutes of Health(AG045781,AG051510,and AG066526)(to WCX).
文摘Wnt/β-catenin signaling is critical for various cellular processes in multiple cell types,including osteoblast(OB)differentiation and function.Exactly how Wnt/β-catenin signaling is regulated in OBs remain elusive.ATP6AP2,an accessory subunit of V-ATPase,plays important roles in multiple cell types/organs and multiple signaling pathways.However,little is known whether and how ATP6AP2 in OBs regulates Wnt/β-catenin signaling and bone formation.Here we provide evidence for ATP6AP2 in the OB-lineage cells to promote OB-mediated bone formation and bone homeostasis selectively in the trabecular bone regions.Conditionally knocking out(CKO)ATP6AP2 in the OB-lineage cells(Atp6ap2^(Ocn-Cre))reduced trabecular,but not cortical,bone formation and bone mass.Proteomic and cellular biochemical studies revealed that LRP6 and N-cadherin were reduced in ATP6AP2-KO BMSCs and OBs,but not osteocytes.Additional in vitro and in vivo studies revealed impairedβ-catenin signaling in ATP6AP2-KO BMSCs and OBs,but not osteocytes,under both basal and Wnt stimulated conditions,although LRP5 was decreased in ATP6AP2-KO osteocytes,but not BMSCs.Further cell biological studies uncovered that osteoblastic ATP6AP2 is not required for Wnt3a suppression ofβ-catenin phosphorylation,but necessary for LRP6/β-catenin and N-cadherin/β-catenin protein complex distribution at the cell membrane,thus preventing their degradation.Expression of activeβ-catenin diminished the OB differentiation deficit in ATP6AP2-KO BMSCs.Taken together,these results support the view for ATP6AP2 as a critical regulator of both LRP6 and N-cadherin protein trafficking and stability,and thus regulatingβ-catenin levels,demonstrating an un-recognized function of osteoblastic ATP6AP2 in promoting Wnt/LRP6/β-catenin signaling and trabecular bone formation.
基金financially supported by the National Natural Science Foundation of China (Grant No. 82270854)the Natural Science Foundation of Chongqing+2 种基金China (No. cstc2020jcyj-msxm X0408)the Program for Youth Innovation in Future MedicineChongqing Medical University (No. W0162) to Yinyuan Ding。
文摘Background: LDL receptor-related protein-1(LRP1) is a cell-surface receptor that functions in diverse physiological pathways. We previously demonstrated that hepatocyte-specific LRP1 deficiency(hLRP1KO) promotes diet-induced insulin resistance and increases hepatic gluconeogenesis in mice. However, it remains unclear whether LRP1 regulates hepatic glycogenesis.Methods: Insulin signaling, glycogenic gene expression, and glycogen content were assessed in mice and HepG2 cells. The pcDNA 3.1 plasmid and adeno-associated virus serotype 8 vector(AAV8) were used to overexpress the truncated β-chain(βΔ) of LRP1 both in vitro and in vivo.Results: On a normal chow diet, hLRP1KO mice exhibited impaired insulin signaling and decreased glycogen content. Moreover, LRP1 expression in HepG2 cells was significantly repressed by palmitate in a dose-and time-dependent manner. Both LRP1 knockdown and palmitate treatment led to reduced phosphorylation of Akt and GSK3β, increased levels of phosphorylated glycogen synthase(GYS), and diminished glycogen synthesis in insulin-stimulated HepG2 cells, which was restored by exogenous expression of the βΔ-chain. By contrast, AAV8-mediated hepatic βΔ-chain overexpression significantly improved the insulin signaling pathway, thus activating glycogenesis and enhancing glycogen storage in the livers of high-fat diet(HFD)-fed mice.Conclusion: Our data revealed that LRP1, especially its β-chain, facilitates hepatic glycogenesis by improving the insulin signaling pathway, suggesting a new therapeutic strategy for hepatic insulin resistance-related diseases.
文摘Objective: To study the multidrug resistance (MDR) mechanism of lung resistance protein (LRP) gene in hepatocellular carcinoma (HCC), and the relations among the expression of the LRP gene and clinicopathologic features, the influence of α-fetoprotein (AFP), and prognosis of patients who received adjuvant chemotherapy after resection of HCC. Methods: The expression of the LRP gene encoding LRP and mRNA LRP was detected in tissues from 54 untreated patients with HCC, adjacent tissues from 24 patients with HCC and archival paraffin-embedded tissues from 12 patients with posthepatitic cirrhosis. The relationship between the LRP gene expression and the change of AFP level was analyzed in the 24 postoperative HCC patients whose AFP was measured after 2 weeks. All of the HCC patients were followed up. Results: The percentage of positive expression of LRP and mRNA LRP in the 3 tissues was 61.1%, 33.3%, 16.7%, and 75.9%, 37.5%, 33.3% respectively. There was significant difference between the untreated HCC tissue and other tissues (P<0.05). No difference existed between the LRP gene expression and clinicopathologic findings, age, sex, and tumor size (P>0.05), but the expression was related to the degree of differentiation of HCC (P<0.05). The effective rate of AFP in the LRP gene positive expression group or in postoperative chemotherapeutic patients was very lower than that in the negative group (P<0.05). Although the mean survival time of postoperative HCC patients in negative LRP gene expression group was longer than that of positive group, there was no difference between them (P<0.05). Conclusion: LRP gene expression is related to MDR of HCC and initiates the intrinsic MDR. Detection of LRP gene expression is of great guiding significance in accessing chemotherapeutic resistance of HCC. As an index to chemotherapy of HCC, detection of LRP expression provides evidence for making individual chemotherapeutic treatment,and reversing MDR in HCC. Although LRP gene expression correlates with the tumor differential degree (P<0.05), it perhaps does not relate with the prognosis of HCC patients.
