蛋白质组学特征分析揭示了人类多能基质细胞中活跃的 Wnt 信号传导是β细胞存活和增殖的关键调节因子。

Proteomic characterisation reveals active Wnt-signalling by human multipotent stromal cells as a key regulator of beta cell survival and proliferation.

机构信息

Don Rix Protein Identification Facility, Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, N5A 6C1, Canada.

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada.

出版信息

Diabetologia. 2017 Oct;60(10):1987-1998. doi: 10.1007/s00125-017-4355-7. Epub 2017 Jul 14.

DOI:10.1007/s00125-017-4355-7

PMID:28710530

Abstract

AIMS/HYPOTHESIS: Novel strategies to stimulate the expansion of beta cell mass in situ are warranted for diabetes therapy. The aim of this study was to elucidate the secretome of human bone marrow (BM)-derived multipotent stromal cells (MSCs) with documented islet regenerative paracrine function. We hypothesised that regenerative MSCs will secrete a unique combination of protein factors that augment islet regeneration.

METHODS

Human BM-derived MSCs were examined for glucose-lowering capacity after transplantation into streptozotocin-treated NOD/severe combined immunodeficiency (SCID) mice and segregated into samples with regenerative (MSC) vs nonregenerative (MSC) capacity. Secreted proteins associated with islet regenerative function were identified using stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomics. To functionally validate the importance of active Wnt signalling, we stimulated the Wnt-signalling pathway in MSC samples during ex vivo expansion using glycogen synthase kinase 3 (GSK3) inhibition (CHIR99201), and the conditioned culture media (CM) generated was tested for the capacity to support cultured human islet cell survival and proliferation in vitro.

RESULTS

MSC showed increased secretion of proteins associated with cell growth, matrix remodelling, immunosuppressive and proangiogenic properties. In contrast, MSC uniquely secreted proteins known to promote inflammation and negatively regulate angiogenesis. Most notably, MSC maintained Wnt signalling via Wnt5A/B (~~2.5-fold increase) autocrine activity during ex vivo culture, while MSC repressed Wnt signalling via Dickkopf-related protein (DKK)1 (~~2.5-fold increase) and DKK3 secretion. Inhibition of GSK3 activity in MSC samples increased the accumulation of nuclear β-catenin and generated CM that augmented beta cell survival (13% increases) and proliferation when exposed to cultured human islets.

CONCLUSIONS/INTERPRETATION: Maintenance of active Wnt signalling within human MSCs promotes the secretion of matricellular and proangiogenic proteins that formulate a niche for islet regeneration.

摘要

目的/假设：刺激原位β细胞群体扩张的新策略是糖尿病治疗所必需的。本研究旨在阐明具有胰岛再生旁分泌功能的人骨髓（BM）来源多能基质细胞（MSC）的分泌组。我们假设再生 MSC 将分泌独特的蛋白因子组合，增强胰岛再生。

方法

在链脲佐菌素处理的 NOD/严重联合免疫缺陷（SCID）小鼠中移植人 BM 来源 MSC 后，检查其降低血糖的能力，并将其分为具有再生（MSC）和非再生（MSC）能力的样本。使用稳定同位素标记与细胞培养中的氨基酸（SILAC）-基于定量蛋白质组学鉴定与胰岛再生功能相关的分泌蛋白。为了功能验证活性 Wnt 信号的重要性，我们在 MSC 样本的体外扩增过程中使用糖原合酶激酶 3（GSK3）抑制剂（CHIR99201）刺激 Wnt 信号通路，并且测试生成的条件培养基（CM）是否具有支持体外培养的人胰岛细胞存活和增殖的能力。

结果

MSC 显示与细胞生长、基质重塑、免疫抑制和促血管生成特性相关的蛋白分泌增加。相比之下，MSC 独特地分泌已知促进炎症和负调节血管生成的蛋白。值得注意的是，MSC 通过 Wnt5A/B（增加约 2.5 倍）自分泌活性在体外培养过程中维持 Wnt 信号，而 MSC 通过 Dickkopf 相关蛋白（DKK）1（增加约 2.5 倍）和 DKK3 分泌抑制 Wnt 信号。在 MSC 样本中抑制 GSK3 活性会增加核 β-连环蛋白的积累，并产生 CM，当暴露于培养的人胰岛时，CM 会增加β细胞的存活（增加 13%）和增殖。

结论/解释：在人 MSC 内维持活跃的 Wnt 信号会促进细胞外基质和促血管生成蛋白的分泌，从而为胰岛再生形成一个龛位。