Baldauf Conny K, Fahldieck Corinna, Angenstein Alexa, Weinert Sönke, Hakobyan Mariam, Lipka Daniel B, Haage Tobias R, Bhuria Vikas, Böttcher Martin, Mougiakakos Dimitrios, Schraven Burkhart, Fischer Thomas
Institute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany.
Healthcampus Immunology, Inflammation and Infectiology (GC-I3), Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany.
Cell Commun Signal. 2025 Aug 11;23(1):368. doi: 10.1186/s12964-025-02358-x.
The JAK2-V617F mutation is the most frequent driver mutation in a group of malignant hematopoietic disorders called myeloproliferative neoplasms (MPN). JAK2-V617F is a somatic mutation originating in a hematopoietic stem cell and results in constitutively activated JAK-STAT signaling. High levels of pro-inflammatory cytokines in the blood are a hallmark of MPN patients and are a key factor in the severe clinical symptoms seen in these patients. The molecular mechanisms underlying the up-regulation of inflammatory cytokines in JAK2-V617F mutated hematopoietic cells remain to be elucidated.
32D myeloid progenitor cells expressing JAK2-wildtype (WT) and JAK2-V617F, respectively were employed. In addition, primary hematopoietic cells from the JAK2-V617F knock-in MPN mouse model were investigated. Integrin outside-in signaling upon binding of cells to the adhesion molecules VCAM-1/ICAM-1 was characterized by Western blotting of phosphorylated FAK, STAT3, p65, SYK and JNK. Regulation of mRNA and protein expression of IL-1α, IL-1β, IL-6, TNF and CXCL10 was measured by qPCR and ELISA. RNAseq and DNA methylation analysis in primary mouse JAK2-V617F granulocytes was performed. In JAK2-V617F knock-in mice, anti-integrin treatment was applied to evaluate the impact of activated integrin signaling on IL-1 blood levels in vivo.
Integrin stimulation via the adhesion molecules VCAM-1/ICAM-1 activated integrin outside-in signaling including FAK, SYK, NFκB, and JNK. This induced strong mRNA expression of IL-1α, IL-1β, IL-6, TNF and CXCL10. In 32D cells, the presence of the JAK2-V617F mutation further increased VCAM-1/ICAM-1-induced mRNA and protein levels of IL-1α and IL-1β, and active caspase 1 expression. In primary granulocytes, integrin stimulation resulted in an activated mRNA signature of inflammatory cytokines. Consistent with the mRNA results, adhesion to VCAM-1/ICAM-1 induced an increase in intracellular IL-1α and IL-1β protein levels in 32D cells. However, in primary hematopoietic cells, up-regulation of inflammatory cytokines was not observed at the protein level in vitro, whereas, in vivo, blocking of integrin binding to VCAM-1/ICAM-1 was sufficient to reduce elevated IL-1α levels in the blood of JAK2-V617F mice.
We conclude that integrin stimulation via the adhesion molecules VCAM-1/ICAM-1 activates integrin outside-in signaling, leading to the up-regulation of pro-inflammatory cytokines in both JAK2-mutated and non-mutated mouse hematopoietic cells.
JAK2-V617F突变是一组称为骨髓增殖性肿瘤（MPN）的恶性造血疾病中最常见的驱动突变。JAK2-V617F是一种起源于造血干细胞的体细胞突变，可导致JAK-STAT信号通路持续激活。血液中促炎细胞因子水平升高是MPN患者的一个标志，也是这些患者出现严重临床症状的关键因素。JAK2-V617F突变的造血细胞中炎症细胞因子上调的分子机制仍有待阐明。
分别使用表达JAK2野生型（WT）和JAK2-V617F的32D骨髓祖细胞。此外，还研究了JAK2-V617F基因敲入MPN小鼠模型的原代造血细胞。通过对磷酸化FAK、STAT3、p65、SYK和JNK进行蛋白质印迹分析，表征细胞与黏附分子VCAM-1/ICAM-1结合时整合素外向内信号传导。通过qPCR和ELISA检测IL-1α、IL-1β、IL-6、TNF和CXCL10的mRNA和蛋白质表达调控。对原代小鼠JAK2-V617F粒细胞进行RNAseq和DNA甲基化分析。在JAK2-V617F基因敲入小鼠中，应用抗整合素治疗来评估激活的整合素信号对体内IL-1血液水平的影响。
通过黏附分子VCAM-1/ICAM-1刺激整合素可激活整合素外向内信号传导，包括FAK、SYK、NFκB和JNK。这诱导了IL-1α、IL-1β、IL-6、TNF和CXCL10的强烈mRNA表达。在32D细胞中，JAK2-V617F突变的存在进一步增加了VCAM-1/ICAM-1诱导的IL-1α和IL-1β的mRNA和蛋白质水平，以及活性半胱天冬酶1的表达。在原代粒细胞中，整合素刺激导致炎症细胞因子的激活mRNA特征。与mRNA结果一致，黏附于VCAM-1/ICAM-1可诱导32D细胞内IL-1α和IL-1β蛋白水平升高。然而，在原代造血细胞中，体外未观察到炎症细胞因子在蛋白质水平上的上调，而在体内，阻断整合素与VCAM-1/ICAM-1的结合足以降低JAK2-V617F小鼠血液中升高的IL-1α水平。
我们得出结论，通过黏附分子VCAM-1/ICAM-1刺激整合素可激活整合素外向内信号传导，导致JAK2突变和未突变的小鼠造血细胞中促炎细胞因子上调。
