Frommherz L H, Sayar S B, Wang Y, Trefzer L K, He Y, Leppert J, Eßer P, Has C
Department of Dermatology, Medical Center - University of Freiburg, Freiburg, Germany.
Department of Dermatology and Allergology, University Hospital, LMU Munich, Germany.
Matrix Biol Plus. 2022 Aug 11;16:100119. doi: 10.1016/j.mbplus.2022.100119. eCollection 2022 Dec.
Integrin α3β1 is a cell adhesion receptor widely expressed in epithelial cells. Pathogenic variants in the gene encoding the integrin α3 subunit lead to a syndrome including interstitial lung disease, nephrotic syndrome, and epidermolysis bullosa (ILNEB). Renal involvement mainly consists of glomerular disease caused by loss of adhesion between podocytes and the glomerular basement membrane. The aim of this study was to characterize the impact of loss of integrin α3 on human podocytes. was stably knocked-out in the human podocyte cell line AB8/13, designated as Podo, and in human proximal tubule epithelial cell line HK2 using the targeted genome editing technique CRISPR/Cas9. Cell clones were characterized by Sanger sequencing, quantitative PCR, Western Blot and immunofluorescence staining. RNASeq of integrin α3 negative cells and controls was performed to identify differential gene expression patterns. Differentiated Podo did not substantially change morphology and adhesion under standard culture conditions, but displayed significantly reduced spreading and adhesion when seed on laminin 511 in serum free medium. Gene expression studies demonstrated a distinct dysregulation of the adhesion network with downregulation of most integrin α3 interaction partners. In agreement with this, biological processes such as "extracellular matrix organization" and "cell differentiation" as well as KEGG pathways such as "ECM-receptor interaction", "focal adhesion" and the "PI3K-Akt signaling pathway" were significantly downregulated in human podocytes lacking the integrin α3 subunit.
整合素α3β1是一种在上皮细胞中广泛表达的细胞粘附受体。编码整合素α3亚基的基因中的致病变异会导致一种综合征，包括间质性肺病、肾病综合征和大疱性表皮松解症（ILNEB）。肾脏受累主要由足细胞与肾小球基底膜之间粘附丧失引起的肾小球疾病组成。本研究的目的是表征整合素α3缺失对人足细胞的影响。使用靶向基因组编辑技术CRISPR/Cas9在人足细胞系AB8/13（命名为Podo）和人近端肾小管上皮细胞系HK2中稳定敲除整合素α3。通过桑格测序、定量PCR、蛋白质免疫印迹和免疫荧光染色对细胞克隆进行表征。对整合素α3阴性细胞和对照进行RNA测序以鉴定差异基因表达模式。在标准培养条件下，分化的Podo细胞形态和粘附没有实质性变化，但在无血清培养基中接种于层粘连蛋白511时，其铺展和粘附显著降低。基因表达研究表明粘附网络存在明显失调，大多数整合素α3相互作用伙伴下调。与此一致的是，在缺乏整合素α3亚基的人足细胞中，“细胞外基质组织”和“细胞分化”等生物学过程以及“ECM-受体相互作用”、“粘着斑”和“PI3K-Akt信号通路”等KEGG途径均显著下调。
