Adverse cardiovascular events are associated with vascular calcification (VC) process, where vascular smooth muscle cells (VSMCs) differentiate into osteoblastic phenotype and deposit hydroxyapatite crystals. Microtubule-associated protein kinesin family member 2C (KIF2C) expression is decreased obviously in VSMC during calcification induction. Accordingly, we investigate the role and potential mechanism of KIF2C on VSMC calcification. The effects of β-glycerophosphate (β-GP)/KIF2C/lumican (LUM) on calcification, calcium content, alkaline phosphatase (ALP) activity, calcification-related markers, Tubulin, the ratio of polymerized (Po) to free (Fr) tubulin, as well as levels of LUM, apolipoprotein B (APOB), and KIF2C were assessed by Alizarin red S staining, calcium assay kit, ALP assay kit, Western blot, immunofluorescence, and quantitative real-time PCR. The interplay between LUM and APOB was estimated using co-immunoprecipitation and immunofluorescence. As a result, β-GP promoted calcification of human VMSCs (HVMSCs) and repressed KIF2C expression. KIF2C overexpression reversed the effect of β-GP on HVSMCs. LUM silencing attenuated β-GP-induced promotion on HVSMC calcification and increased KIF2C expression by interacting with APOB. Collectively, LUM silencing can alleviate β-GP-induced VSMC calcification through mitigating the repression of APOB on KIF2C expression.
Zhenqiang Li,Chengfeng Sun,Maosong Chen,Boding Wang
A recent study has reported that lumican (LUM) is expressed at a high level in the nucleus pulposus specimens from herniated lumbar disc, without description of the specific mechanism. This study was designed to investigate the function and mechanism of LUM in intervertebral disc degeneration (IDD). In this study, human nucleus pulposus cells (hNPCs) cells were challenged with tumor necrosis factor (TNF)-α to establish the IDD in vitro model. After LUM silencing, cell viability was detected using CCK-8 kit, and the expression of inflammatory factors was evaluated using RT-qPCR and ELISA. Flow cytometry and β-galactosidase staining were used to determine cell cycle and cell senescence. The expression of cycle and senescence-related proteins was evaluated with western blotting. Then, Fas ligand (FasL) was overexpressed and proteins in apoptosis signal regulating kinase 1 (ASK1)/p38 signaling were tested. Finally, GS-4997, an inhibitor of ASK1, was used to explore the regulatory effects of LUM on ASK1/p38 signaling in TNF-α-induced hNPCs. Results indicated that LUM expression was upregulated in TNF-α-challenged hNPCs. LUM gene interference mitigated TNF-α-induced inflammatory response, cell cycle arrest, and senescence of hNPCs. It was then found that LUM silencing could inhibit ASK1/p38 signaling in TNF-α-treated hNPCs, which was reversed by FasL overexpression. Additionally, ASK1/p38 participated in the mediation by LUM of TNF-α-induced inflammation, cell cycle arrest, and senescence of hNPCs. To conclude, interference with LUM effectively mitigated TNF-α-induced inflammatory response, cell cycle arrest, and cell senescence. Further experiments showed the involvement of ASK1/p38 pathway in LUM-mediated NP cell phenotypes through FasL.
Zhang Qinyao,Huang Shuwei,Liu Xiaoqian,Zhang Meijiu,Sun Jun,Liu Mingzhu,Nie Hemin,Lei Lei
Aim During the tooth-root elongation phase, dental epithelial stem cells (DESCs) cluster together and release various signaling molecules, facilitating the differentiation of stem cells from the apical papilla (SCAPs) into odontoblasts. This study aimed to investigate the regulatory mechanism of secretory proteins from DESCs cultured in a spherical system mimicking the in vivo microenvironment on the odontoblastic differentiation of SCAPs during root development, providing theoretical foundations and potential targets for dentin regeneration therapy based on epithelial-mesenchymal interactions. Methodology DESCs were isolated from the root tips of rodent incisors and cultured using low-attachment surface to mimic their in vivo clustering. The effects of spheroid culture (versus monolayer culture) on SCAPs viability and odontoblastic differentiation potential were systematically compared. Label-free mass spectrometry and bioinformatics analyses were employed to characterize the secretory protein profiles of DESCs under different culture conditions. In vitro and in vivo experiments validated the role of a key differential protein Lumican and its downstream signaling mechanisms. Results Compared to monolayer culture, spheroid culture significantly enhanced the secretion of Lumican by DESCs. Lumican demonstrated the ability to significantly promote dentin differentiation of SCAPs in vitro and in vivo. Mechanistic analysis revealed that Lumican binds specifically to integrin β1 (ITGβ1) on the SCAPs membrane, thereby activating the downstream ERK signaling pathway to mediate its pro-differentiation effects. Conclusion This study illustrates that spheroid culture can restore the epithelial microenvironment of DESCs, leading to the release of Lumican, which targets SCAPs. This research uncovers the crucial roles of Lumican and ITGβ1 in DESCs-SACPs interactions and paves the way for potential therapeutic strategies in dentin regeneration. Graphical abstract
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