Quantitative Proteome Analysis of Alveolar Type-II Cells Reveals a Connection of Integrin Receptor Subunits Beta 2/6 and WNT Signaling

Alveolar type-II cells (ATII cells) are lung progenitor cells responsible
for regeneration of alveolar epithelium during homeostatic turnover
and in response to injury. Characterization of ATII cells will have
a profound impact on our understanding and treatment of lung disease.
The identification of novel ATII cell-surface proteins can be used
for sorting and enrichment of these cells for further characterization.
Here we combined a high-resolution mass spectrometry-based membrane
proteomic approach using lungs of the SILAC mice with an Affymetrix
microarray-based transcriptome analysis of ATII cells. We identified
16 proteins that are enriched in the membrane fraction of ATII cells
and whose genes are highly expressed in these cells. Interestingly,
we confirmed our data for two of these genes, integrin beta 2 and
6 (Itgb2 and Itgb6), by qRT-PCR
expression analysis and Western blot analysis of protein extracts.
Moreover, flow cytometry and immunohistochemistry in adult lung revealed
that ITGB2 and ITGB6 are present in subpopulations of surfactant-associated-protein-C-positive
cells, suggesting the existence of different types of ATII cells.
Furthermore, analysis of the Itgb2–/– mice showed that Itgb2 is required for proper WNT
signaling regulation in the lung.

肺泡II型细胞（Alveolar type-II cells，ATII细胞）是肺祖细胞，负责在稳态更新与损伤应答过程中实现肺泡上皮的再生。对ATII细胞的精准解析，将对我们认知与治疗肺部疾病产生深远影响。鉴定新型ATII细胞表面蛋白，可用于这类细胞的分选与富集，以开展后续的表征研究。本研究结合了基于高分辨率质谱的膜蛋白质组学方法（以SILAC小鼠的肺组织为样本）与ATII细胞的Affymetrix芯片转录组分析，共鉴定出16种在ATII细胞膜组分中富集、且其编码基因在该类细胞中高表达的蛋白质。值得注意的是，我们通过蛋白质提取物的实时定量逆转录聚合酶链反应（qRT-PCR）表达分析与蛋白质免疫印迹（Western blot）分析，验证了其中两个基因——整合素β2与β6（Itgb2与Itgb6）的相关数据。此外，通过对成年肺组织进行流式细胞术与免疫组化分析，我们发现ITGB2与ITGB6表达于表面活性蛋白C阳性细胞的亚群中，这提示ATII细胞存在不同的亚型。进一步对Itgb2基因敲除小鼠的分析显示，Itgb2对肺部正常的WNT信号通路调控具有不可或缺的作用。