JOURNAL OF DENTAL RESEARCH
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2020
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IF 4.914
J. Fu,H. Zheng,Y. Xue,R. Jin,G. Yang,Z. Chen,G. Yuan
WW domain-containing E3 Ub-protein ligase 2 (WWP2) belongs to the homologous to E6AP C-terminus (HECT) E3 ligase family. It has been explored to regulate osteogenic differentiation, chondrogenesis, and palatogenesis. Odontoblasts are terminally differentiated mesenchymal cells, which contribute to dentin formation in tooth development. However, it remained unknown whether WWP2 participated in odontoblast differentiation. In this study, WWP2 was found to be expressed in mouse dental papilla cells (mDPCs), odontoblasts, and odontoblastic-induced mDPCs by immunohistochemistry and Western blotting. Besides, WWP2 expression was decreased in the cytoplasm but increased in the nuclei of differentiation-induced mDPCs. When Wwp2 was knocked down, the elevated expression of odontoblast marker genes (Dmp1 and Dspp) in mDPCs induced by differentiation medium was suppressed. Meanwhile, a decrease of alkaline phosphatase (ALP) activity was observed by ALP staining, and reduced formation of mineralized matrix nodules was demonstrated by Alizarin Red S staining. Overexpression of WWP2 presented opposite results to knockdown experiments, suggesting that WWP2 promoted odontoblastic differentiation of mDPCs. Further investigation found that WWP2 was coexpressed and interacted with KLF5 in the nuclei, leading to ubiquitination of KLF5. The PPPSY (PY2) motif of KLF5 was essential for its physical binding with WWP2. Also, cysteine 838 (Cys838) of WWP2 was the active site for ubiquitination of KLF5, which did not lead to proteolysis of KLF5. Then, KLF5 was confirmed to be monoubiquitinated and transactivated by WWP2, which promoted the expression of KLF5 downstream genes Dmp1 and Dspp. Deletion of the PY2 motif of KLF5 or mutation of Cys838 of WWP2 reduced the upregulation of Dmp1 and Dspp. Besides, lysine (K) residues K31, K52, K83, and K265 of KLF5 were verified to be crucial to WWP2-mediated KLF5 transactivation. Taken together, WWP2 promoted odontoblastic differentiation by monoubiquitinating KLF5.
研究领域:
分子生物学
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发育生物学
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细胞生物学
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DOI
PubMed
高影响因子
Frontiers in Physiology
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2021
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IF 4.566
Wangzhou Kaixin,Lai Zhiying,Lu Zishao,Fu Wanren,Liu Cheng,Liang Zhengeng,Tan Yi,Li Conghui,Hao Chunbo
Human periodontal ligament cells (hPDLCs) play a vital role in cell regeneration and tissue repair with multi-directional differentiation potential. microRNAs (miRs) are implicated in the osteogenesis of hPDLCs. This study explored the mechanism of miR-143-3p in osteogenesis of hPDLCs. Osteogenic differentiation of isolated hPDLCs was induced. KLF5 expression during osteogenic differentiation of hPDLCs was detected and then silenced in hPDLCs. Binding relationship between KLF5 and miR-143-3p was predicted and verified. hPDLCs were treated with miR-143-3p mimic or overexpressing KLF5, and then osteogenic specific markers and mineralized nodules were measured. The key factors of the Wnt/β-catenin pathway during osteogenesis of hPDLCs were measured. KLF5 expression was upregulated during osteogenesis of hPDLCs. KLF5 silencing or miR-143-3p mimic reduced osteogenic specific markers and mineralized nodules. Overexpression of KLF5 could reverse the inhibitory effect of miR-143-3p on osteogenic differentiation. miR-143-3p mimic and KLF5 silencing inactivated the Wnt/β-catenin pathway. Activation of the Wnt/β-catenin pathway reversed the repression effect of miR-143-3p mimic on osteogenesis of hPDLCs. In conclusion, miR-143-3p inhibited osteogenic differentiation of hPDLCs by targeting KLF5 and inactivating the Wnt/β-catenin pathway.
研究领域:
牙科学
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分子生物学
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细胞生物学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF BIOLOGICAL CHEMISTRY
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2022
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IF 5.486
Jing Fu,Xiaobo Zhang,Huiwen Zheng,Guobin Yang,Zhi Chen,Guohua Yuan
WW domain–containing E3 Ubiquitin-protein ligase 2 (WWP2) has been found to positively regulate odontoblastic differentiation by monoubiquitinating the transcription factor Kruppel-like factor 5 (KLF5) in a cell culture system. However, the in vivo role of WWP2 in mouse teeth remains unknown. To explore this, here we generated Wwp2 knockout (Wwp2 KO) mice. We found that molars in Wwp2 KO mice exhibited thinner dentin, widened predentin, and reduced numbers of dentinal tubules. In addition, expression of the odontoblast differentiation markers Dspp and Dmp1 was decreased in the odontoblast layers of Wwp2 KO mice. These findings demonstrate that WWP2 may facilitate odontoblast differentiation and dentinogenesis. Furthermore, we show for the first time that phosphatase and tensin homolog (PTEN), a tumor suppressor, is expressed in dental papilla cells and odontoblasts of mouse molars and acts as a negative regulator of odontoblastic differentiation. Further investigation indicated that PTEN is targeted by WWP2 for degradation during odontoblastic differentiation. We demonstrate PTEN physically interacts with and inhibits the transcriptional activity of KLF5 on Dspp and Dmp1. Finally, we found WWP2 was able to suppress the interaction between PTEN and KLF5, which diminished the inhibition effect of PTEN on KLF5. Taken together, this study confirms the essential role of WWP2 and the WWP2–PTEN–KLF5 signaling axis in odontoblast differentiation and dentinogenesis in vivo .
研究领域:
分子生物学
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发育生物学
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牙科科学
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DOI
PubMed
高影响因子
BRITISH JOURNAL OF PHARMACOLOGY
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2024
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IF 6.8
Huilin Liu,Chao Huang,Zhenni Liu,Yuhan Li,Yanan Zhu,Min Gao,Jing Chen,Hui Zhang,Zhengtao Xiao,Wei Zhao
Background and Purpose Cardiac glycosides (CGs), traditionally prescribed for heart failure and arrhythmias, show anticancer potential. However, their mechanisms for preferential inhibition of tumour tissue and constituent malignant cells are not fully elucidated. This study aims to elucidate the therapeutic benefits of CGs in targeting specific tumours and dissect their multi-targeting mechanisms that confer their cytotoxicity against malignant cells. Experimental Approach We designed an integrated workflow to identify therapeutic CGs with high toxicity to certain cancers, investigating their multi-target effects, assessing their toxicity to malignant cells and analysing the prognostic relevance of CGs' target genes. The computational findings were confirmed through gene knockdown, cell viability assays, reactive oxygen species (ROS) measurements and so forth. Key Results CGs modulate multiple genes crucial for ion homeostasis, oxidative stress and apoptosis, with a particularly strong inhibitory effects on uveal melanoma (UVM). Notably, digitoxin suppresses UVM cell proliferation and induces ROS levels by simultaneously targeting STAT3 and KLF5. Single-cell transcriptomic analysis revealed that malignant cells are likely more vulnerable to CGs due to their higher expression of CG target genes compared with surrounding cells in the UVM microenvironment. Conclusions and Implications Given UVM's limited options, our study highlights the potential of digitoxin as a promising novel therapeutic agent for this aggressive and rare ocular cancer. Our comprehensive approach is effective in identifying the potent, cancer-specific therapeutic agents from herbal plants.
研究领域:
肿瘤学
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药理学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL
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2025
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IF 1.7
Wang Ying,Liu Jiahui,Jiang Yiqun,Liu Yanzuo,Kuang Hongying,Feng Xiaoling
Polycystic ovary syndrome (PCOS), a common endocrine disorder affecting over 10% of women, is characterized by hyperandrogenism and ovarian dysfunction. While linked to chronic inflammation and granulosa cell apoptosis, its molecular mechanisms remain unclear. In this study, we elucidated the novel role of Krüppel-like factor 5 (KLF5) in the pathogenesis of PCOS and its regulatory role with thioredoxin interacting protein (TXNIP). In a mouse model of PCOS induced by dehydroepiandrosterone (DHEA), KLF5 expression was significantly elevated in ovarian tissues (up-regulated 2.62-fold, P < 0.001), correlating with hyperandrogenism (testosterone: up-regulated 2.83-fold, P < 0.001) and cystic follicle formation. The proliferative capacity of testosterone-treated KGN cells was reduced to 59% after KLF5 was knocked down ( P < 0.01), attenuated apoptosis by inhibiting the increase of Bax and Cleaved-caspase 3 proteins and thus attenuated inflammation by down-regulating NLRP3 and Interleukin- (IL-) 1β. Most importantly, dual luciferase assay showed that KLF5 transcriptionally activated TXNIP, resulting in a 3.04-fold enhancement of its promoter activity ( P < 0.001). Meanwhile, overexpression of TXNIP reversed the silencing effect of KLF5, resulting in a significant increase in apoptosis and secretion of inflammatory factors. These results reveal a previously unrecognized KLF5/TXNIP axis driving granulosa cell (GC) dysfunction in polycystic ovary syndrome, in which KLF5 transcriptionally upregulates TXNIP to promote apoptosis and NLRP3 inflammasome activation. Our findings provide the first evidence linking KLF5 to the pathogenesis of PCOS and establish this pathway as a potential therapeutic target, bridging a significant gap in understanding the molecular basis of the disease and providing compelling evidence for clinical drug development.
研究领域:
生殖内分泌学
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分子生物学
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病理生理学
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转化医学
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妇科疾病
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产品关联
DOI
PubMed
高影响因子
LABORATORY INVESTIGATION
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2023
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IF 5
Xiao Li,Doudou Chen,Bowen Ouyang,Shengnan Wang,Yawei Li,Li Li,Siquan Zhu,Guangying Zheng
Diabetic cataract (DC) is a common cause of visual loss in older diabetic subjects. Krüppel-like factor 5 (KLF5) plays an essential role in migration and the epithelial-mesenchymal transition (EMT) in diverse cells and is involved in oxidative stress. However, the effects of KLF5 on DC remain unknown. This study aimed to examine the biological function of KLF5 in DC and its underlying mechanism. The expression patterns of KLF5 were detected in vivo and in vitro . Then KLF5 was knocked down in HLECs to explore its function roles and underlying mechanisms. Dual-luciferase reporter assay and chromatin immunoprecipitation analysis were used to detect whether KLF5 could bind the promoter of MDM2, a key regulator of EMT. Lastly, the regulation of KLF5 in the biological behaviors of HLECs via MDM2 was analyzed. We found a significant increase in KLF5 in the DC lens anterior capsular, diabetic rat lens, and HG-stimulated HLECs. Knockdown of KLF5 inhibited oxidative stress, inflammation, migration, and EMT of HG-stimulated HLECs. KLF5 silencing impeded MDM2 expression and restricted the activation of MARK1/FAK and NF-κB signaling pathways in HLECs under HG condition. Additionally, KLF5 was found to bind the MDM2 promoter and enhance the transcriptional activity of MDM2. The protective effects by silencing KLF5 on HG-cultured HLECs could be offset by MDM2 overexpression. We demonstrated that knockdown of KLF5 alleviated oxidative stress, migration, and EMT of HG-cultured HLECs by regulating MDM2, suggesting a potential therapeutic strategy for DC.
研究领域:
眼科学
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细胞生物学
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分子生物学
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糖尿病研究
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DOI
PubMed
高影响因子
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