Proteomic Analysis of Tumor Necrosis Factor-α-Induced Secretome of Human Adipose Tissue-Derived Mesenchymal Stem Cells

Abstract

Human adipose tissue-derived mesenchymal stem cells (hASCs) are useful for regeneration of inflamed or injured tissues. To identify secreted hASC proteins during inflammation, hASCs were exposed to tumor necrosis factor-alpha (TNF-alpha) and conditioned media derived from hASCs were analyzed by liquid chromatography coupled with tandem mass spectrometry. We identified 187 individual proteins as secreted proteins (secretome) in hASC-conditioned media; 118 proteins were secreted at higher levels upon TNF-alpha treatment. The TNF-alpha-induced secretome included a variety of cytokines and chemokines such as interleukin-6 (IL-6), IL-8, chemokine (C-X-C motif) ligand 6, and monocyte chemotactic protein-1 (MCP-1). TNF-alpha also increased expression of various proteases including cathepsin L, matrix metalloproteases and protease inhibitors, and induced secretion of long pentraxin 3, a key inflammatory mediator implicated in innate immunity. TNF-alpha-conditioned media stimulated migration of human monocytes, which play a key role in inflammatory responses. This migration was abrogated by pretreatment with neutralizing anti-IL-6, anti-IL-8, and anti-MCP-1 antibodies, suggesting that IL-6, IL-8, and MCP-1 are involved in migration of monocytes. Taken together, these results suggest that TNF-alpha-induced secretome may play a pivotal role in inflammatory responses and that shotgun proteomic analysis will be useful for elucidation of the paracrine functions of mesenchymal stem cells.

SH3BGRL3 Protein as a Potential Prognostic Biomarker for Urothelial Carcinoma: A Novel Binding Partner of Epidermal Growth Factor Receptor

“…2C). A recent study reported that SH3BGRL3 is upregulated in the secretome of human adipose tissue-derived mesenchymal stem cells upon TNFa treatment (18). SH3BGRL3 was classified as a nonclassical secretory protein ( Supplementary Fig.…”

Plasma PTX3 protein levels inversely correlate with insulin secretion and obesity, whereas visceral adipose tissue PTX3 gene expression is increased in obesity

“…Furthermore, we corroborate that marked upregulation of PTX3 gene expression and protein content by IL-1␤ and TNF␣ in cultured SGBS adipocytes is parallel to increased protein secretion. Indeed, secretion of PTX3 protein from different stimulated cell types has been demonstrated, e.g., in rodent 3T3-F442A adipocytes (1), AT-derived mesenchymal stem cells (25), monocytes, endothelial cells and fibroblasts (3,20), myoblasts (20), FS4 fibroblasts, and Hep3B hepatocytes (23). In conclusion, our data in cultured adipocytes suggest that some proinflammatory factors elevated in AT in obesity such as IL-1␤ (22) and TNF␣ (19) may contribute to the upregulation of PTX3 production in the VAT depots of obese subjects, whereas other obesityrelated factors such as AT oxidative stress and hypoxia or hyperinsulinemia do not substantially regulate PTX3 gene expression in adipocytes.…”

“…Indeed, PTX3 has different cell-specific gene expression and ligand-binding properties from CRP (8). PTX3 is produced in response to proinflammatory signals by mononuclear phagocytes (3,20), myeloid dendritic cells (12), fibroblasts (9,20,23), endothelial cells (9,20), hepatic cells (9), myoblasts (20), synoviocytes (26), lung epithelial cells (17), renal epithelial cells (32), adipocytes (1), AT-derived mesenchymal stem cells (25), and smooth muscle cells (11). Expression of the PTX3 gene increases in human cultured myotubes compared with the skeletal muscle tissue (38).…”

Differential Response of Human Adipose Tissue-Derived Mesenchymal Stem Cells, Dermal Fibroblasts, and Keratinocytes to Burn Wound Exudates: Potential Role of Skin-Specific Chemokine CCL27

“…cell type: ASC, 31 fibroblasts, 32 and keratinocytes 21 10 ng/mL TNF-a (R&D Systems); granulocytes 10 nM N-Formylmethionyl-leucyl-phenylalanine (fMLP) (GenScript, Piscataway, NJ) 33 ; monocytes 100 ng/mL Lipopolysaccharides (LPS) (Sigma-Aldrich) 34 ; or HMVEC 10 ng/mL vascular endothelial growth factor (VEGF) (PrepoTech, Rocky Hill, NJ). 18 Culture supernatants were harvested (granulocytes after 4 h, monocytes after 16 h, and ASC, fibroblasts, keratinocytes, and HMVEC after 24 h) and stored at -20°C for further analysis by ELISA.…”