Abstract
Mesenchymal stem cells (MSCs) secrete cytokines in a paracrine or autocrine manner to regulate immune response and tissue regeneration. Our previous research revealed that MSCs use the complex of Fas/Fas-associated phosphatase-1 (Fap-1)/caveolin-1 (Cav-1) mediated exocytotic process to regulate cytokine and small extracellular vesicles (EVs) secretion, which contributes to accelerated wound healing. However, the detailed underlying mechanism of cytokine secretion controlled by Cav-1 remains to be explored. We show that Gingiva-derived MSCs (GMSCs) could secrete more C-X-C motif chemokine ligand 10 (CXCL10) but showed lower phospho-Cav-1 (p-Cav-1) expression than skin-derived MSCs (SMSCs). Moreover, dephosphorylation of Cav-1 by a Src kinase inhibitor PP2 significantly enhances CXCL10 secretion, while activating phosphorylation of Cav-1 by H2O2 restraints CXCL10 secretion in GMSCs. We also found that Fas and Fap-1 contribute to the dephosphorylation of Cav-1 to elevate CXCL10 secretion. Tumor necrosis factor-α serves as an activator to up-regulate Fas, Fap-1, and down-regulate p-Cav-1 expression to promote CXCL10 release. Furthermore, local applying p-Cav-1 inhibitor PP2 could accelerate wound healing, reduce the expression of α-smooth muscle actin and increase cleaved-caspase 3 expression. These results indicated that dephosphorylation of Cav-1 could inhibit fibrosis during wound healing. The present study establishes a previously unknown role of p-Cav-1 in controlling cytokine release of MSC and may present a potential therapeutic approach for promoting scarless wound healing.
Highlights
MATERIALS AND METHODSWound healing is a complex process involving increased proliferation, adhesion, and migration of cells from connective tissue and epithelium, inflammatory reactions, and extracellular matrix remodeling
We found that in addition to previously reported interleukin-1 receptor antagonist (IL-1RA) (Kou et al, 2018), Gingiva-derived MSCs (GMSCs) secreted a higher amount of CXCL10, a potent chemokine for activated T lymphocytes, compared to skin-derived MSCs (SMSCs) (Figure 1A)
To test whether tumor necrosis factor-α (TNF-α) affects Fas/Fas-associated phosphatase-1 (Fap-1) controlled Cav-1 dephosphorylation and Cav-1 dephosphorylation-related CXCL10 secretion, we showed that TNF-α treatment upregulated the expression of Fas or Fap-1 but not Cav-1 in WT GMSC, MRL/lpr GMSCs, and Fap-1 small interfering RNA (siRNA)-treated GMSCs (Figures 6B,C), which was consistent with our previous study (Kou et al, 2018)
Summary
Wound healing is a complex process involving increased proliferation, adhesion, and migration of cells from connective tissue and epithelium, inflammatory reactions, and extracellular matrix remodeling. Our previous study showed that GMSCs displayed different secretion profiles compared with skin-derived MSCs (SMSCs) and produce a higher amount of interleukin-1 receptor antagonist (IL-1RA) to accelerate gingival and cutaneous wound healing in mice (Kou et al, 2018). Anti-CXCL10 (sc-374092), Cav-1 (7C8) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Murine GMSCs and SMSCs were isolated and cultured as reported by our previous study (Zhang et al, 2009; Xu et al, 2013; Kou et al, 2018) To put it gingival tissue around the molars and dorsal skin tissues of mice were separated and minced. Proteins were quantified using the BCA protein assay kit (Thermo Scientific, United States) following the manufacturer’s instructions.
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