Limits...
LL-37 stimulates the functions of adipose-derived stromal/stem cells via early growth response 1 and the MAPK pathway.

Yang Y, Choi H, Seon M, Cho D, Bang SI - Stem Cell Res Ther (2016)

Bottom Line: To examine the effects of LL-37 on ASC function, cell proliferation and migration were measured by a cell counting kit (CCK-8) and a Transwell migration assay.Activation of mitogen-activated protein kinases (MAPKs) was essential not only for LL-37-enhanced ASC proliferation and migration but also EGR1 expression; treatment with a specific inhibitor of extracellular signal-regulated kinase, p38, or c-Jun N-terminal kinase blocked the stimulatory effect of LL-37.This study correlates LL-37 with MSC functions (specifically those of ASCs), including cell expansion, cell migration, and paracrine actions, which may be useful in terms of implantation for tissue regeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Plastic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.

ABSTRACT

Background: LL-37 is a naturally occurring antimicrobial peptide found in the wound bed and assists wound repair. No published study has characterized the role of LL-37 in the function(s) of human mesenchymal stem cells (MSCs). This study investigated the functions of adipose-derived stromal/stem cells (ASCs) activated by LL-37 by performing both in vitro assays with cultured cells and in vivo assays with C57BL/6 mice with hair loss.

Methods: Human ASCs were isolated from healthy donors with written informed consent. To examine the effects of LL-37 on ASC function, cell proliferation and migration were measured by a cell counting kit (CCK-8) and a Transwell migration assay. Early growth response 1 (EGR1) mRNA expression was determined by microarray and real-time PCR analyses. The protein levels of EGR1 and regenerative factors were analyzed by specific enzyme-linked immunosorbent assays and western blotting.

Results: LL-37 treatment enhanced the proliferation and migration of human ASCs expressing formyl peptide receptor like-1. Microarray and real-time PCR data showed that EGR1 expression was rapidly and significantly increased by LL-37 treatment. LL-37 treatment also enhanced the production of EGR1. Moreover, small interfering RNA-mediated knockdown of EGR1 inhibited LL-37-enhanced ASC proliferation and migration. Activation of mitogen-activated protein kinases (MAPKs) was essential not only for LL-37-enhanced ASC proliferation and migration but also EGR1 expression; treatment with a specific inhibitor of extracellular signal-regulated kinase, p38, or c-Jun N-terminal kinase blocked the stimulatory effect of LL-37. EGR1 has a strong paracrine capability and can influence angiogenic factors in ASCs; therefore, we evaluated the secretion levels of vascular endothelial growth factor, thymosin beta-4, monocyte chemoattractant protein-1, and stromal cell-derived factor-1. LL-37 treatment increased the secretion of these regenerative factors. Moreover, treatment with the conditioned medium of ASCs pre-activated with LL-37 strongly promoted hair growth in vivo.

Conclusions: These findings show that LL-37 increases EGR1 expression and MAPK activation, and that preconditioning of ASCs with LL-37 has a strong potential to promote hair growth in vivo. This study correlates LL-37 with MSC functions (specifically those of ASCs), including cell expansion, cell migration, and paracrine actions, which may be useful in terms of implantation for tissue regeneration.

No MeSH data available.


Related in: MedlinePlus

Effects of LL-37 on the proliferation and migration of human ASCs. a FPRL1 expression on ASCs from four donors, as analyzed by flow cytometry. b Proliferating cells were measured at 24 and 48 hr using the CCK-8 assay. A representative experiment of three independent experiments is shown. Bars represent the mean ± SD. *, P < 0.01 vs. control at 24 or 48 hr. c Immunostaining was performed and visualized using a confocal microscope. Bar = 100 μm. d ASCs were seeded in the upper wells and the lower wells were treated with 5, 10, and 20 μg/mL LL-37 for 6 hr. A migration assay was performed using Transwell chambers. Cells that migrated were counted using Scanscope (images show samples treated with 20 μg/mL LL-37). A representative experiment from three independent experiments is shown. Bars represent the mean ± SEM. *, P < 0.05 vs. control. e ASC proliferation was analyzed after pretreatment of cells with Ptx or an anti-LL-37 neutralizing antibody (αLL-37) with or without LL-37. f ASC migration was analyzed after pretreatment of cells with Ptx or αLL-37 prior to LL-37 treatment. Values represent the mean ± SD of three independent experiments. *, P < 0.01 vs. control; §, P < 0.01 vs. LL-37-treated cells. ASCs adipose-derived stromal/stem cells, FPRL1 formyl peptide receptor like-1, Ptx pertussis toxin
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4837546&req=5

Fig1: Effects of LL-37 on the proliferation and migration of human ASCs. a FPRL1 expression on ASCs from four donors, as analyzed by flow cytometry. b Proliferating cells were measured at 24 and 48 hr using the CCK-8 assay. A representative experiment of three independent experiments is shown. Bars represent the mean ± SD. *, P < 0.01 vs. control at 24 or 48 hr. c Immunostaining was performed and visualized using a confocal microscope. Bar = 100 μm. d ASCs were seeded in the upper wells and the lower wells were treated with 5, 10, and 20 μg/mL LL-37 for 6 hr. A migration assay was performed using Transwell chambers. Cells that migrated were counted using Scanscope (images show samples treated with 20 μg/mL LL-37). A representative experiment from three independent experiments is shown. Bars represent the mean ± SEM. *, P < 0.05 vs. control. e ASC proliferation was analyzed after pretreatment of cells with Ptx or an anti-LL-37 neutralizing antibody (αLL-37) with or without LL-37. f ASC migration was analyzed after pretreatment of cells with Ptx or αLL-37 prior to LL-37 treatment. Values represent the mean ± SD of three independent experiments. *, P < 0.01 vs. control; §, P < 0.01 vs. LL-37-treated cells. ASCs adipose-derived stromal/stem cells, FPRL1 formyl peptide receptor like-1, Ptx pertussis toxin

Mentions: LL-37 reportedly activates functions such as proliferation and migration through FPRL1, a G-protein-coupled receptor, in many cell types [18]; therefore, several donor pools of ASCs were examined for expression of FPRL1. Flow cytometric data confirmed that FPRL1 was expressed on ASCs in each donor pool (Fig. 1a).Fig. 1


LL-37 stimulates the functions of adipose-derived stromal/stem cells via early growth response 1 and the MAPK pathway.

Yang Y, Choi H, Seon M, Cho D, Bang SI - Stem Cell Res Ther (2016)

Effects of LL-37 on the proliferation and migration of human ASCs. a FPRL1 expression on ASCs from four donors, as analyzed by flow cytometry. b Proliferating cells were measured at 24 and 48 hr using the CCK-8 assay. A representative experiment of three independent experiments is shown. Bars represent the mean ± SD. *, P < 0.01 vs. control at 24 or 48 hr. c Immunostaining was performed and visualized using a confocal microscope. Bar = 100 μm. d ASCs were seeded in the upper wells and the lower wells were treated with 5, 10, and 20 μg/mL LL-37 for 6 hr. A migration assay was performed using Transwell chambers. Cells that migrated were counted using Scanscope (images show samples treated with 20 μg/mL LL-37). A representative experiment from three independent experiments is shown. Bars represent the mean ± SEM. *, P < 0.05 vs. control. e ASC proliferation was analyzed after pretreatment of cells with Ptx or an anti-LL-37 neutralizing antibody (αLL-37) with or without LL-37. f ASC migration was analyzed after pretreatment of cells with Ptx or αLL-37 prior to LL-37 treatment. Values represent the mean ± SD of three independent experiments. *, P < 0.01 vs. control; §, P < 0.01 vs. LL-37-treated cells. ASCs adipose-derived stromal/stem cells, FPRL1 formyl peptide receptor like-1, Ptx pertussis toxin
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4837546&req=5

Fig1: Effects of LL-37 on the proliferation and migration of human ASCs. a FPRL1 expression on ASCs from four donors, as analyzed by flow cytometry. b Proliferating cells were measured at 24 and 48 hr using the CCK-8 assay. A representative experiment of three independent experiments is shown. Bars represent the mean ± SD. *, P < 0.01 vs. control at 24 or 48 hr. c Immunostaining was performed and visualized using a confocal microscope. Bar = 100 μm. d ASCs were seeded in the upper wells and the lower wells were treated with 5, 10, and 20 μg/mL LL-37 for 6 hr. A migration assay was performed using Transwell chambers. Cells that migrated were counted using Scanscope (images show samples treated with 20 μg/mL LL-37). A representative experiment from three independent experiments is shown. Bars represent the mean ± SEM. *, P < 0.05 vs. control. e ASC proliferation was analyzed after pretreatment of cells with Ptx or an anti-LL-37 neutralizing antibody (αLL-37) with or without LL-37. f ASC migration was analyzed after pretreatment of cells with Ptx or αLL-37 prior to LL-37 treatment. Values represent the mean ± SD of three independent experiments. *, P < 0.01 vs. control; §, P < 0.01 vs. LL-37-treated cells. ASCs adipose-derived stromal/stem cells, FPRL1 formyl peptide receptor like-1, Ptx pertussis toxin
Mentions: LL-37 reportedly activates functions such as proliferation and migration through FPRL1, a G-protein-coupled receptor, in many cell types [18]; therefore, several donor pools of ASCs were examined for expression of FPRL1. Flow cytometric data confirmed that FPRL1 was expressed on ASCs in each donor pool (Fig. 1a).Fig. 1

Bottom Line: To examine the effects of LL-37 on ASC function, cell proliferation and migration were measured by a cell counting kit (CCK-8) and a Transwell migration assay.Activation of mitogen-activated protein kinases (MAPKs) was essential not only for LL-37-enhanced ASC proliferation and migration but also EGR1 expression; treatment with a specific inhibitor of extracellular signal-regulated kinase, p38, or c-Jun N-terminal kinase blocked the stimulatory effect of LL-37.This study correlates LL-37 with MSC functions (specifically those of ASCs), including cell expansion, cell migration, and paracrine actions, which may be useful in terms of implantation for tissue regeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Plastic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.

ABSTRACT

Background: LL-37 is a naturally occurring antimicrobial peptide found in the wound bed and assists wound repair. No published study has characterized the role of LL-37 in the function(s) of human mesenchymal stem cells (MSCs). This study investigated the functions of adipose-derived stromal/stem cells (ASCs) activated by LL-37 by performing both in vitro assays with cultured cells and in vivo assays with C57BL/6 mice with hair loss.

Methods: Human ASCs were isolated from healthy donors with written informed consent. To examine the effects of LL-37 on ASC function, cell proliferation and migration were measured by a cell counting kit (CCK-8) and a Transwell migration assay. Early growth response 1 (EGR1) mRNA expression was determined by microarray and real-time PCR analyses. The protein levels of EGR1 and regenerative factors were analyzed by specific enzyme-linked immunosorbent assays and western blotting.

Results: LL-37 treatment enhanced the proliferation and migration of human ASCs expressing formyl peptide receptor like-1. Microarray and real-time PCR data showed that EGR1 expression was rapidly and significantly increased by LL-37 treatment. LL-37 treatment also enhanced the production of EGR1. Moreover, small interfering RNA-mediated knockdown of EGR1 inhibited LL-37-enhanced ASC proliferation and migration. Activation of mitogen-activated protein kinases (MAPKs) was essential not only for LL-37-enhanced ASC proliferation and migration but also EGR1 expression; treatment with a specific inhibitor of extracellular signal-regulated kinase, p38, or c-Jun N-terminal kinase blocked the stimulatory effect of LL-37. EGR1 has a strong paracrine capability and can influence angiogenic factors in ASCs; therefore, we evaluated the secretion levels of vascular endothelial growth factor, thymosin beta-4, monocyte chemoattractant protein-1, and stromal cell-derived factor-1. LL-37 treatment increased the secretion of these regenerative factors. Moreover, treatment with the conditioned medium of ASCs pre-activated with LL-37 strongly promoted hair growth in vivo.

Conclusions: These findings show that LL-37 increases EGR1 expression and MAPK activation, and that preconditioning of ASCs with LL-37 has a strong potential to promote hair growth in vivo. This study correlates LL-37 with MSC functions (specifically those of ASCs), including cell expansion, cell migration, and paracrine actions, which may be useful in terms of implantation for tissue regeneration.

No MeSH data available.


Related in: MedlinePlus