Kim Ji Hyun, Irfan Muhammad, Sreekumar Sreelekshmi, Chong Kerwin, Hong Jin, Alapati Satish, Chung Seung
Department of Oral Biology, College of Dentistry, University of Illinois Chicago, Chicago, IL, United States.
Department of Endodontics, College of Dentistry, University of Illinois Chicago, Chicago, IL, United States.
Front Cell Dev Biol. 2025 Mar 26;13:1558736. doi: 10.3389/fcell.2025.1558736. eCollection 2025.
CaMKII is a serine/threonine-specific protein kinase that plays a crucial role in normal and pathological conditions. However, limited information is available regarding the roles of CaMKII in dentinogenesis, particularly in an inflammatory context. Previously, we demonstrated the pivotal role of TrkB in inflammation-induced differentiation of hDPSCs into odontoblast-like cells. Here, we investigate the interaction between CaMKII and TrkB during hDPSCs odontogenic differentiation. hDPSCs were cultured and subjected to CaMKII knockdown using siRNA, followed by treatment with dentinogenic media. TNFα-stimulated cells were treated with CaMKII- inhibitor, -protein, or TrkB antagonist, CTX-B. Immunocytochemistry and ARS were used to visualize targeted proteins and calcium deposits. Real-time PCR detected expression levels of odontogenic and mineralization markers such as DSPP and DMP-1. Our data indicate that CaMKII inhibition enhances TrkB protein levels and promotes TNFα-induced transcriptional activation of genes associated with odontogenic differentiation. CaMKII knockdown via siRNA and pharmacological inhibition elevated DSPP and DMP-1 protein levels, whereas CaMKII overexpression suppressed their expression. Notably, treatment with TNF-α and a CaMKII inhibitor upregulated DSPP and DMP-1 expression, while co-treatment with CTX-B abolished this effect. Similarly, mRNA expression of DSPP and DMP-1 was reduced at day 10. Mineralization activity exhibited a similar pattern to the expression of these markers. Our findings unveil a novel mechanism underlying the role of CaMKII via TrkB in dentinogenesis, which is vital for the success of hDPSCs engineering strategies.
钙调蛋白激酶II（CaMKII）是一种丝氨酸/苏氨酸特异性蛋白激酶，在正常和病理条件下均发挥着关键作用。然而，关于CaMKII在牙本质形成中的作用，尤其是在炎症背景下的作用，目前可用信息有限。此前，我们证明了酪氨酸激酶B（TrkB）在炎症诱导人牙髓干细胞（hDPSCs）向成牙本质细胞样细胞分化过程中的关键作用。在此，我们研究hDPSCs牙源性分化过程中CaMKII与TrkB之间的相互作用。培养hDPSCs，并用小干扰RNA（siRNA）敲低CaMKII，随后用牙源性培养基处理。用CaMKII抑制剂、CaMKII蛋白或TrkB拮抗剂霍乱毒素B亚基（CTX - B）处理肿瘤坏死因子α（TNFα）刺激的细胞。采用免疫细胞化学和茜素红染色（ARS）观察靶向蛋白和钙沉积情况。实时聚合酶链反应（PCR）检测牙源性和矿化标志物如牙本质涎磷蛋白（DSPP）和牙本质基质蛋白1（DMP - 1）的表达水平。我们的数据表明，抑制CaMKII可提高TrkB蛋白水平，并促进TNFα诱导的与牙源性分化相关基因的转录激活。通过siRNA敲低CaMKII以及药物抑制均可提高DSPP和DMP - 1蛋白水平，而CaMKII过表达则抑制它们的表达。值得注意的是，用TNF - α和CaMKII抑制剂处理可上调DSPP和DMP - 1的表达，而与CTX - B共同处理则消除了这种作用。同样，在第10天时DSPP和DMP - 1的信使核糖核酸（mRNA）表达降低。矿化活性与这些标志物的表达呈现相似模式。我们的研究结果揭示了CaMKII通过TrkB在牙本质形成中发挥作用的一种新机制，这对hDPSCs工程策略的成功至关重要。
