Dephosphorylation of Caveolin-1 Controls C-X-C Motif Chemokine Ligand 10 Secretion in Mesenchymal Stem Cells to Regulate the Process of Wound Healing


Posted: 2021-11-18 20:00:00
Front Cell Dev Biol . 2021 Nov 1;9:725630. doi: 10.3389/fcell.2021.725630. eCollection 2021. Affiliations Expand Affiliations 1 South China Center of Craniofacial Stem Cell Research, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China. 2 Department of Microbiology, Zhongshan School of Medicine, Key Laboratory for Tropical Diseases Control of the Ministry of Education, Sun Yat-sen University, Guangzhou, China. 3 Key Laboratory for Stem Cells and Tissue Engineering, Sun Yat-sen University, Ministry of Education, Guangzhou, China. Item in Clipboard Panpan Wang et al. Front Cell Dev Biol. 2021. Show details Display options Display options Format Front Cell Dev Biol . 2021 Nov 1;9:725630. doi: 10.3389/fcell.2021.725630. eCollection 2021. Affiliations 1 South China Center of Craniofacial Stem Cell Research, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China. 2 Department of Microbiology, Zhongshan School of Medicine, Key Laboratory for Tropical Diseases Control of the Ministry of Education, Sun Yat-sen University, Guangzhou, China. 3 Key Laboratory for Stem Cells and Tissue Engineering, Sun Yat-sen University, Ministry of Education, Guangzhou, China. Item in Clipboard CiteDisplay options Display options Format 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. Keywords: CXCL10; mesenchymal stem cells; phospho-caveolin-1; secretion; wound healing. Copyright © 2021 Wang, Zhao, Wang, Wu, Sui, Mao, Shi and Kou. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Figures FIGURE 1 Murine GMSCs produce and secrete… FIGURE 1 Murine GMSCs produce and secrete higher amounts of CXCL10 than SMSCs. (A) Cytokine… FIGURE 1 Murine GMSCs produce and secrete higher amounts of CXCL10 than SMSCs. (A) Cytokine array analysis of cytokines in cell culture supernatant from mouse GMSCs and SMSCs. Equal volume of cell culture supernatant were taken from 48 h incubation of 0.4 × 106 cells. (B) ELISA analysis of CXCL10 in the culture supernatant of GMSCs and SMSCs. (C) Western blotting analysis showed that the protein expression of CXCL10 was higher in GMSCs than SMSCs. β-Actin was used as a protein loading control. (D) CXCL10 (green) co-expressed with the MSC marker CD73 (red) in GMSCs and SMSCs as analyzed by immunocytofluorescence staining. Scale bar, 20 μm. All results are representative of data obtained from at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars are means ± SD. Data were analyzed using independent unpaired two-tailed Student’s t-tests. FIGURE 2 Murine GMSCs displayed lower Cav-1… FIGURE 2 Murine GMSCs displayed lower Cav-1 phosphorylation than SMSCs. (A) Western blotting analysis showed… FIGURE 2 Murine GMSCs displayed lower Cav-1 phosphorylation than SMSCs. (A) Western blotting analysis showed the expression of p-Cav-1 and Cav-1 in GMSCs and SMSCs. (B) Higher resolution images of Cav-1 (red) and p-Cav-1 (green) in MSCs captured by SIM microscopy. Scale bar, 20 μm. Right panel, 3D reconstruction of 2D Z-stack data showed the different steric structures of p-Cav-1 in GMSCs and SMSCs. Scale bar, 2 μm. (C) Immunofluorescence analysis showed that p-Cav-1 or Cav-1 (green) co-localized with the MSC marker CD73 (red) in GMSCs. Scale bar, 200 μm. (D) Representative immunofluorescence staining images of p-Cav-1 and Cav-1 (green) in GMSCs at indicated time point after seeding to the culture plate. Time dependent p-Cav-1 translocation could be observed. Scale bar, 50 μm. FIGURE 3 Dephosphorylation of Cav-1 controlled CXCL10… FIGURE 3 Dephosphorylation of Cav-1 controlled CXCL10 secretion in murine GMSCs. (A) Western blotting analysis… FIGURE 3 Dephosphorylation of Cav-1 controlled CXCL10 secretion in murine GMSCs. (A) Western blotting analysis showed cytoplasmic protein expression of p-Cav-1, Cav-1, and CXCL10 of GMSCs treated with different dose of PP2 for 2 h. (B) ELISA analysis of CXCL10 secretion in the culture supernatant from GMSCs treated with different dose of PP2 for 24 h. *P < 0.05 compared with control group. (C) Western blotting analysis showed cytoplasmic protein expression of p-Cav-1, Cav-1, and CXCL10 of GMSCs treated with different dose of H2O2 for 0.5 h. (D) ELISA analysis of CXCL10 secretion in the culture supernatant from GMSCs treated with different dose of H2O2 for 24 h. **P < 0.01 compared with control group. (E) Western blotting analysis showed absent expression of Cav-1 and p-Cav-1 and decreased cytoplasmic CXCL10 in Cav-1–/– GMSCs. (F) Knocking out of Cav-1 elevated CXCL10 secretion into the culture supernatant of GMSCs as analyzed by ELISA. ***P < 0.001 compared with WT GMSCs control. (G) Western blotting analysis confirmed the efficiency of Cav-1 siRNA, and showed that knocking down of Cav-1 decreased cytoplasmic CXCL10 in GMSCs. (H) Knocking down of Cav-1 elevated CXCL10 secretion in the culture supernatant of GMSCs as analyzed by ELISA. **P < 0.01 compared with GMSCs treated with control siRNA. All results are representative of data generated in at least three independent experiments. Error bars are means ± SD. Data were analyzed using independent unpaired two-tailed Student’s t-tests or one-way ANOVA. FIGURE 4 Fas controlled dephosphorylation of Cav-1… FIGURE 4 Fas controlled dephosphorylation of Cav-1 and CXCL10 release in murine GMSCs. (A) Western… FIGURE 4 Fas controlled dephosphorylation of Cav-1 and CXCL10 release in murine GMSCs. (A) Western blotting analysis of p-Cav-1, Cav-1, and CXCL10 in GMSCs from Fas-deficient MRL/lpr mice. (B) Knocking out of Fas down-regulated CXCL10 secretion in GMSCs culture supernatant as analyzed by ELISA. ***P < 0.001 compared with WT GMSCs control. (C) Western blotting analysis confirmed the efficiency of Fas siRNA, and showed that knocking down of Fas up-regulated p-Cav-1 expression and reduced cytoplasmic CXCL10 in GMSCs. (D) Knocking down of Fas decreased CXCL10 secretion in the culture supernatant of GMSCs as analyzed by ELISA. *P < 0.05 compared with GMSCs treated with control siRNA. (E) Higher resolution images of Cav-1 (red) and p-Cav-1 (green) in MSCs captured by SIM microscopy. Scale bar, 20 μm. Right panel, 3D reconstruction of 2D Z-stack data showed the different steric structures of p-Cav-1 in GMSCs and MRL/Lpr GMSCs. Scale bar, 2 μm. (F) Immunocytofluorescence staining images of p-Cav-1, Cav-1, (green) and Fas (red) in GMSCs. Scale bar, 20 μm. All results are representative of data generated in at least three independent experiments. Error bars are means ± SD. Data were analyzed using independent unpaired two-tailed Student’s t-tests. FIGURE 5 Fas-associated phosphatase-1 controlled dephosphorylation of… FIGURE 5 Fas-associated phosphatase-1 controlled dephosphorylation of Cav-1 and CXCL10 release in murine GMSCs. (A)… FIGURE 5 Fas-associated phosphatase-1 controlled dephosphorylation of Cav-1 and CXCL10 release in murine GMSCs. (A) Western blotting analysis was used to confirm the efficiency of Fap-1 siRNA, and knocking down of Fap-1 up-regulated p-Cav-1 expression but reduced cytoplasmic CXCL10. (B) ELISA analysis showed that knocking down of Fap-1 decreased CXCL10 secretion in the culture supernatant of GMSCs. *P < 0.05. (C) Immunocytofluorescence staining images of p-Cav-1 (green) and Fap-1 (red) in WT control and Fap-1 deficient GMSCs. Scale bar, 20 μm. (D) Immunocytofluorescence staining images of Cav-1 (green) and Fap-1 (red) in WT control and Fap-1 deficient GMSCs. Scale bar, 20 μm. All results are representative of data generated in at least three independent experiments. Error bars are means ± SD. Data were analyzed using independent unpaired two-tailed Student’s t-tests. FIGURE 6 Tumor necrosis factor-αTNF-α up-regulates Fas… FIGURE 6 Tumor necrosis factor-αTNF-α up-regulates Fas and Fap-1 to dephosphorylate Cav-1 to activate CXCL10… FIGURE 6 Tumor necrosis factor-αTNF-α up-regulates Fas and Fap-1 to dephosphorylate Cav-1 to activate CXCL10 secretion in murine GMSCs. (A) ELISA analysis of CXCL10 secretion in the culture supernatant from WT GMSCs treated with indicated dose of TNF-α or IFN-γ. ***P < 0.001, TNF-α or IFN-γ treated GMSCs versus control; #P < 0.05, ##P < 0.01, TNF-α treated GMSCs versus IFN-γ treated GMSCs. (B) Western blotting analysis of Fap-1, Fas, p-Cav-1, Cav-1, CXCL10 protein expression of WT GMSCs and MRL/Lpr GMSCs with or without TNF-α (20 ng/ml) treatment. (C) Western blotting analysis of Fap-1, Fas, p-Cav-1, Cav-1, CXCL10 expression of control and Fap-1 siRNA-treated GMSCs with or without TNF-α (20 ng/ml) treatment. (D) ELISA analysis of CXCL10 secretion in the culture supernatant from WT GMSCs, MRL/lpr GMSCs with or without TNF-α (20 ng/ml) treatment for 48 h. **P < 0.01, ***P < 0.001. (E) ELISA analysis of CXCL10 secretion in the culture supernatant from control and Fap-1 siRNA-treated GMSCs with or without TNF-α (20 ng/ml) treatment for 48 h. *P < 0.05*, **P < 0.01, ***P < 0.001. (F) Immunocytofluorescence staining of GMSCs at various time points after TNF-α (20 ng/ml) treatment. p-Cav-1 (green) translocation from the cell membrane to the cytoplasm was indicated. Scale bar, 20 μm. (G) Western blotting analysis of protein expression of p-Cav-1, Cav-1 in cell membrane and cytoplasm. The results confirmed the translocation of p-Cav-1 from the cell membrane to cytoplasm upon TNF-α treatment. All results are representative of data generated in at least three independent experiments. Error bars are means ± SD. Data were analyzed using independent unpaired two-tailed Student’s t-tests. FIGURE 7 Dephosphorylation of caveolin-1 controls wound… FIGURE 7 Dephosphorylation of caveolin-1 controls wound healing process in mice. (A) Scheme diagram illustrating… FIGURE 7 Dephosphorylation of caveolin-1 controls wound healing process in mice. (A) Scheme diagram illustrating the cutaneous wound procedure in C57BL/6 mice and treatment with PP2 or PBS. (B) Immunocytofluorescence staining of p-Cav-1 (green) and CXCL10 (red) in the wound area in control and PP2 treated group. Scale bar, 100 μm. Magnified images of the boxed region are showed in the upper right boxes. (C) Representative macroscopic images of cutaneous wound area in mice after treatment with and without PP2. (D) Quantification of wound area in mice over time. PP2 treatment could accelerate wound healing process on 9 and 11 days post-wounding compared to WT control mice. *P < 0.05, **P < 0.01. (E) Representative H&E image from cutaneous wounds in control and PP2 treated mice 14 days post-wounding. Scale bar, 500 μm. The black dash lines indicate the margin the unhealed wound area. The right box is a higher magnification of the boxed region in the image. Scale bar, 20 μm. (F) Evaluation of collagen maturity by Masson’s trichrome staining of wounds following treatment with PBS or PP2 at 14 days post-wounding. Scale bar, 500 μm. The right box is a higher magnification of the boxed region in the image. Scale bar, 50 μm. Note the bulky collagen fibers in control group. (G) Immunofluorescence analysis showed the expression of Collagen I and Collagen III (green) in the wound area in control and PP2 treated group. Scale bar, 100 μm. (H) Immunofluorescence analysis showed the staining of α-SMA (red) and PCNA (green) in the wound area in control and PP2 treated group. Scale bar, 100 μm. (I) TUNEL-positive apoptotic cells (red) in the wound area of PP2 treated group was significantly higher than in the control group. Scale bar, 20 μm. (J) Western blotting analysis of protein expression of α-SMA, PCNA, caspase 3, and cleaved-caspase 3 in wound area. All figures (7) References Abe Y., Murano M., Murano N., Morita E., Inoue T., Kawakami K., et al. (2012). Simvastatin attenuates intestinal fibrosis independent of the anti-inflammatory effect by promoting fibroblast/myofibroblast apoptosis in the regeneration/healing process from TNBS-induced colitis. Dig. Dis. Sci. 57 335–344. 10.1007/s10620-011-1879-4 - DOI - PubMed Bernardo M. E., Fibbe W. E. (2013). Mesenchymal stromal cells: sensors and switchers of inflammation. Cell Stem Cell 13 392–402. 10.1016/j.stem.2013.09.006 - DOI - PubMed Bodnar R. J., Yates C. C., Rodgers M. E., Du X., Wells A. (2009). IP-10 induces dissociation of newly formed blood vessels. J. Cell Sci. 122(Pt 12), 2064–2077. 10.1242/jcs.048793 - DOI - PMC - PubMed Buwa N., Kannan N., Kanade S., Balasubramanian N. (2021). Adhesion-dependent Caveolin-1 tyrosine-14 phosphorylation is regulated by FAK in response to changing matrix stiffness. FEBS Lett. 595 532–547. 10.1002/1873-3468.14025 - DOI - PubMed Chen D. B., Li S. M., Qian X. X., Moon C., Zheng J. (2005). Tyrosine phosphorylation of caveolin 1 by oxidative stress is reversible and dependent on the c-src tyrosine kinase but not mitogen-activated protein kinase pathways in placental artery endothelial cells. Biol. Reprod. 73 761–772. 10.1095/biolreprod.105.040881 - DOI - PubMed Show all 46 references [x] Cite Copy Format: Send To [x]

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