Knockout of NOS2 Promotes Adipogenic Differentiation of Rat MSCs by Enhancing Activation of JAK/STAT3 Signaling.

Aiping Qin,Ling-Ran Du,Yu Zhang,Xiyong Yu,Andy Peng Xiang,Li Ding,Lu Liang,De-Hua Lai,Sheng Chen,Ping Wang,Xiaotao Huang

doi:10.3389/fcell.2021.638518

Abstract

Mesenchymal stromal cells (MSCs) are a heterogeneous population of cells that possess multilineage differentiation potential and extensive immunomodulatory properties. In mice and rats, MSCs produce nitric oxide (NO), as immunomodulatory effector molecule that exerts an antiproliferative effect on T cells, while the role of NO in differentiation was less clear. Here, we investigated the role of NO synthase 2 (NOS2) on adipogenic and osteogenic differentiation of rat MSCs. MSCs isolated from NOS2-null (NOS2–/–) and wild type (WT) Sprague–Dawley (SD) rats exhibited homogenous fibroblast-like morphology and characteristic phenotypes. However, after induction, adipogenic differentiation was found significantly promoted in NOS2–/– MSCs compared to WT MSCs, but not in osteogenic differentiation. Accordingly, qRT-PCR revealed that the adipogenesis-related genes PPAR-γ, C/EBP-α, LPL and FABP4 were markedly upregulated in NOS2–/– MSCs, but not for osteogenic transcription factors or marker genes. Further investigations revealed that the significant enhancement of adipogenic differentiation in NOS2–/– MSCs was due to overactivation of the STAT3 signaling pathway. Both AG490 and S3I-201, small molecule inhibitors that selectively inhibit STAT3 activation, reversed this adipogenic effect. Furthermore, after high-fat diet (HFD) feeding, knockout of NOS2 in rat MSCs resulted in significant obesity. In summary, NOS2 is involved in the regulation of rat MSC adipogenic differentiation via the STAT3 signaling pathway.

Highlights

Mesenchymal stromal cells (MSCs) are self-renewing multipotent stromal cells that can be isolated from mesenchymal tissues, such as bone marrow, adipose tissue, dental pulp, umbilical cord blood, and other tissues
Flow cytometry analysis revealed that bone marrow-derived MSCs (BMSCs) from both wild type (WT) and NO synthase 2 (NOS2)−/−rats expressed the same panel of surface markers, including CD29 and CD90, but not the hematopoietic stem cell markers CD34 or CD45 (Figure 1B), indicating that NOS2 knockout may not alter the phenotype of BMSCs
Knockout of rat NOS2 did not alter the proliferative properties of BMSCs, which were verified by Cell Counting Kit-8 (CCK8) assays (p = 0.49, Figure 1C)

Summary

Introduction

Mesenchymal stromal cells (MSCs) are self-renewing multipotent stromal cells that can be isolated from mesenchymal tissues, such as bone marrow, adipose tissue, dental pulp, umbilical cord blood, and other tissues. The therapeutic potential of MSCs is attributed to complex cellular and molecular mechanisms of action, including regulation of immune responses via immunomodulation and differentiation into multiple cell lineages. MSCs are capable of differentiating into cartilage, bone, tendon, adipose tissue, muscle, etc. Among these tissues, MSCs are commonly progenitors of osteoblasts and adipocytes (Pittenger et al, 1999). The precise mechanisms that regulate the differentiation of MSCs and determine the fate of stem cells are still unclear (Pittenger et al, 1999; Discher et al, 2009; Chen et al, 2016; Li et al, 2019)

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Conclusion