Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.
Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.
Cell Death Dis. 2022 May 20;13(5):481. doi: 10.1038/s41419-022-04932-4.
Bone marrow (BM) fibrosis was thought to be induced exclusively by mesenchymal stromal cells (MSCs). However, we and others found that neoplastic fibrocytes induce BM fibrosis in myelofibrosis (MF). Because glioma-associated oncogene-1 (GLI1), an effector of the Hedgehog pathway, plays a role in the induction of BM fibrosis, we wondered whether GLI1 affects fibrocyte-induced BM fibrosis in MF. Multiplexed fluorescence immunohistochemistry analysis of MF patients' BM detected high levels of GLI1 in MF fibrocytes compared to MSCs or normal fibrocytes. Immunostaining, RNA in situ hybridization, gene expression analysis, and western immunoblotting detected high levels of GLI1 and GLI1-induced matrix metalloproteases (MMP) 2 and 9 in MF patients BM-derived cultured fibrocytes. Similarly, MF patients' BM-derived GLI1 fibrocytes were found in BMs and spleens of MF xenograft mice. GLI1 silencing reduced the levels of MMP2/9, phosphorylated SMAD2/3, and procollagen-I, and knockdown or inhibition of GLI1 decreased fibrocyte formation and induced apoptosis of both fibrocytes and fibrocyte progenitors. Because Janus kinase (JAK)2-induced STAT3 is constitutively activated in MF and because STAT3 induces GLI1 expression, we sought to determine whether STAT3 activates GLI1 in MF fibrocytes. Imaging analysis detected phosphotyrosine STAT3 in MF patients' BM fibrocytes, and transfection of fibrocytes with STAT3-siRNA or treatment with a JAK1/2 inhibitor ruxolitinib reduced GLI1 and MMP2/9 levels. Chromatin immunoprecipitation and a luciferase assay revealed that STAT3 induced the expression of the GLI1 gene in both MF BM fibrocytes and fibrocyte progenitors. Together, our data suggest that STAT3-activated GLI1 contributes to the induction of BM fibrosis in MF.
骨髓（BM）纤维化被认为是由间充质基质细胞（MSCs）引起的。然而，我们和其他人发现，肿瘤性成纤维细胞在骨髓纤维化（MF）中诱导 BM 纤维化。由于 Hedgehog 通路的效应物Glioma-associated oncogene-1（GLI1）在诱导 BM 纤维化中起作用，我们想知道 GLI1 是否影响 MF 中纤维母细胞诱导的 BM 纤维化。MF 患者 BM 的多重荧光免疫组织化学分析检测到，与 MSCs 或正常纤维母细胞相比，MF 纤维母细胞中的 GLI1 水平较高。免疫染色、RNA 原位杂交、基因表达分析和 Western 免疫印迹检测到 MF 患者 BM 来源培养的纤维母细胞中高水平的 GLI1 和 GLI1 诱导的基质金属蛋白酶（MMP）2 和 9。同样，在 MF 异种移植小鼠的 BM 和脾脏中也发现了 MF 患者 BM 来源的 GLI1 纤维母细胞。GLI1 沉默降低了 MMP2/9、磷酸化 SMAD2/3 和前胶原-I 的水平，GLI1 的敲低或抑制减少了纤维母细胞的形成，并诱导了纤维母细胞和纤维母细胞前体的凋亡。由于在 MF 中 Janus 激酶（JAK）2 诱导的 STAT3 持续激活，并且 STAT3 诱导 GLI1 的表达，我们试图确定 STAT3 是否在 MF 纤维母细胞中激活 GLI1。成像分析检测到 MF 患者 BM 纤维母细胞中的磷酸化酪氨酸 STAT3，纤维母细胞的 STAT3-siRNA 转染或 JAK1/2 抑制剂 ruxolitinib 的处理降低了 GLI1 和 MMP2/9 的水平。染色质免疫沉淀和荧光素酶测定显示 STAT3 诱导 MF BM 纤维母细胞和纤维母细胞前体中 GLI1 基因的表达。总之，我们的数据表明，STAT3 激活的 GLI1 有助于诱导 MF 中的 BM 纤维化。
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