Prolyl endopeptidase-mediated transcriptional regulation in quiescent and activated bone marrow-derived macrophages (Bulk RNA-seq)

Liver fibrosis, a pathogical change commonly existing in various chronic liver diseases, can ultimately develop into life-threatening liver cirrhosis, but effective antifibrotic therapy is absent due to limited understanding of fibrogenic process. The characteristic fibrotic scar in fibrosis is stiff and disorganized extracellular matrix (ECM) yielded by ECM remodeling, a pathogenic process with vigorous participation of aberrantly activated macrophages. Prolyl endopeptidase (PREP) is a dipeptidyl peptidase that possess both proteolytic and non-proteolytic functions. Using Prep knockout mouse, we found aggravated liver fibrosis in a NASH-related fibrosis murine model, as well as significantly altered transcriptome by in vitro M1/M2 polarization in primary macrophages after PREP ablation. Most importantly, a PREP-mediated direct transcriptional regulation to active cis-regulatory genomic regions was identified for the first time as a novel non-proteolytic function of PREP, based on significant nuclear localization of PREP and CUT&Tag-seq results. Among the PREP-downstream genes, Ctsb and Ctsd, genes encoding ECM remodeling-related profibrogenic cathepsin B and D respectively, were excessively expressed in PREP-ablated macrophages in vitro and in vivo. Our results indicates that PREP is a newly found transcriptional regulator in macrophage, and is a promising therapeutic target for fibrosis-related aberrant macrophage activation. Overall design: Bone marrow cells isolated from femora of Prep-/- and Prep+/- littermates were cultivated under 50 ng/ml M-CSF for 7 days to obtain fully differentiated bone marrow-derived macrophages (BMDMs). Then BMDMs were left unstimulated (M0), or stimulated with 50ng/ml LPS (M1), or stimulated with 20 ng/ml recombinant murine Interleukin-4 and 20 ng/ml recombinant murine Interleukin-13 (M2) for 24 hours. Total mRNA of BMDMs was extracted and subjected to RNA-seq.

肝纤维化是多种慢性肝病中普遍存在的病理改变，最终可进展为危及生命的肝硬化，但由于对纤维化发生过程的认知不足，目前尚无有效的抗纤维化治疗手段。纤维化特征性的纤维瘢痕是细胞外基质（extracellular matrix, ECM）重塑产生的僵硬且紊乱的细胞外基质，该致病过程伴随异常活化的巨噬细胞的广泛参与。脯氨酰内肽酶（Prolyl endopeptidase, PREP）是一种兼具蛋白水解与非蛋白水解功能的二肽基肽酶。通过使用Prep基因敲除小鼠，我们在非酒精性脂肪性肝炎（non-alcoholic steatohepatitis, NASH）相关纤维化小鼠模型中观察到肝纤维化加重；同时，在体外原代巨噬细胞中敲除PREP后，M1/M2极化过程中的转录组发生显著改变。最为重要的是，基于PREP显著的核定位现象以及CUT&Tag测序（CUT&Tag-seq）结果，我们首次发现PREP可直接调控活性顺式调控基因组区域的转录，这是PREP全新的非蛋白水解功能。在PREP的下游基因中，组织蛋白酶B（cathepsin B, Ctsb）与组织蛋白酶D（cathepsin D, Ctsd）分别编码与细胞外基质重塑相关的促纤维化组织蛋白酶，二者在体外及体内PREP敲除的巨噬细胞中均呈现过度表达。我们的研究结果表明，PREP是巨噬细胞中全新发现的转录调控因子，有望成为纤维化相关异常巨噬细胞活化的治疗靶点。总体实验设计：从Prep基因敲除（Prep-/-）与同窝野生型杂合（Prep+/-）小鼠的股骨中分离骨髓细胞，在含50 ng/ml巨噬细胞集落刺激因子（macrophage colony-stimulating factor, M-CSF）的培养基中培养7天，以获得完全分化的骨髓来源巨噬细胞（bone marrow-derived macrophages, BMDMs）。随后将BMDMs分为三组：不作刺激（M0型）、用50 ng/ml脂多糖（lipopolysaccharide, LPS）刺激（M1型）、或用20 ng/ml重组小鼠白细胞介素-4与20 ng/ml重组小鼠白细胞介素-13刺激（M2型），刺激时长均为24小时。提取BMDMs的总mRNA并进行RNA测序（RNA-seq）。