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

EGR1 is critical for human LL-37-enhanced ASC migration and proliferation. a EGR1 mRNA expression was determined by real-time PCR. EGR1 mRNA was detected after treatment with 10 and 20 μg/mL LL-37 for 0–6 hr. b ASCs were incubated with 20 μg/mL LL-37 for 0–6 hr. Cell lysates were collected for western blot analysis. The EGR1 protein level was increased by LL-37 treatment in a time-dependent manner. c ASCs were treated with LL-37 and transfected with siRNA targeting EGR1 or a negative control sequence. After stabilization, cells were collected and lysed by the addition of 200 μL of cell lysis buffer. EGR1-targeting siRNA transfection was quantified by performing an EGR1-specific ELISA of cell lysates. d Cells that migrated after EGR1-targeting siRNA transfection were imaged by microscopy and counted using Scanscope. Results are expressed as the mean ± SD of three independent experiments. *, P < 0.01 vs. control; §, P < 0.01 vs. LL-37-treated cells transfected with negative control siRNA. e The effects on human ASC proliferation were similar to those on migration. A representative experiment from three independent experiments is shown. Bars represent the mean ± SD. *, P < 0.01 vs. control; §, P < 0.01 vs. LL-37-treated cells transfected with negative control siRNA. EGR1 early growth response 1, ASC adipose-derived stromal/stem cells, siRNA small interfering RNA, ELISA enzyme-linked immunosorbent assay
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: EGR1 is critical for human LL-37-enhanced ASC migration and proliferation. a EGR1 mRNA expression was determined by real-time PCR. EGR1 mRNA was detected after treatment with 10 and 20 μg/mL LL-37 for 0–6 hr. b ASCs were incubated with 20 μg/mL LL-37 for 0–6 hr. Cell lysates were collected for western blot analysis. The EGR1 protein level was increased by LL-37 treatment in a time-dependent manner. c ASCs were treated with LL-37 and transfected with siRNA targeting EGR1 or a negative control sequence. After stabilization, cells were collected and lysed by the addition of 200 μL of cell lysis buffer. EGR1-targeting siRNA transfection was quantified by performing an EGR1-specific ELISA of cell lysates. d Cells that migrated after EGR1-targeting siRNA transfection were imaged by microscopy and counted using Scanscope. Results are expressed as the mean ± SD of three independent experiments. *, P < 0.01 vs. control; §, P < 0.01 vs. LL-37-treated cells transfected with negative control siRNA. e The effects on human ASC proliferation were similar to those on migration. A representative experiment from three independent experiments is shown. Bars represent the mean ± SD. *, P < 0.01 vs. control; §, P < 0.01 vs. LL-37-treated cells transfected with negative control siRNA. EGR1 early growth response 1, ASC adipose-derived stromal/stem cells, siRNA small interfering RNA, ELISA enzyme-linked immunosorbent assay

Mentions: We investigated the target genes of LL-37 that induce ASC proliferation and migration using the Agilent human 4x44K array, a human signaling pathway finder. Microarray analysis showed that LL-37 treatment significantly increased EGR1 expression by 18.47-fold (Table 1 and Additional file 3: Table S2). LL-37 treatment also increased the levels of several genes (including EGR2, early growth response 2; KLF10, Krupple-like factor 10; FOS; and CTGF, connective tissue growth factor) linked to various functions, such as the cell cycle, cell migration, cell proliferation, and transcription. To confirm the effect of LL-37 treatment on EGR1 expression in ASCs, cells were treated with 10 or 20 μg/mL LL-37 for different amounts of time and then real-time PCR analysis was performed. LL-37 treatment significantly increased the EGR1 mRNA level, which peaked at 1 hr and returned to the basal level at about 6 hr (Fig. 2a). Because LL-37 treatment considerably increased the mRNA level of EGR1, we attempted to ascertain whether it also increased the protein level of EGR1 via western blot analysis. LL-37 treatment rapidly increased the protein level of EGR1 in ASCs in a time-dependent manner (Fig. 2b). In addition to western blot analysis, ELISA analysis of cell lysates demonstrated that EGR1 production was significantly increased by LL-37 treatment (Additional file 2: Figure S1b)Table 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)

EGR1 is critical for human LL-37-enhanced ASC migration and proliferation. a EGR1 mRNA expression was determined by real-time PCR. EGR1 mRNA was detected after treatment with 10 and 20 μg/mL LL-37 for 0–6 hr. b ASCs were incubated with 20 μg/mL LL-37 for 0–6 hr. Cell lysates were collected for western blot analysis. The EGR1 protein level was increased by LL-37 treatment in a time-dependent manner. c ASCs were treated with LL-37 and transfected with siRNA targeting EGR1 or a negative control sequence. After stabilization, cells were collected and lysed by the addition of 200 μL of cell lysis buffer. EGR1-targeting siRNA transfection was quantified by performing an EGR1-specific ELISA of cell lysates. d Cells that migrated after EGR1-targeting siRNA transfection were imaged by microscopy and counted using Scanscope. Results are expressed as the mean ± SD of three independent experiments. *, P < 0.01 vs. control; §, P < 0.01 vs. LL-37-treated cells transfected with negative control siRNA. e The effects on human ASC proliferation were similar to those on migration. A representative experiment from three independent experiments is shown. Bars represent the mean ± SD. *, P < 0.01 vs. control; §, P < 0.01 vs. LL-37-treated cells transfected with negative control siRNA. EGR1 early growth response 1, ASC adipose-derived stromal/stem cells, siRNA small interfering RNA, ELISA enzyme-linked immunosorbent assay
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: EGR1 is critical for human LL-37-enhanced ASC migration and proliferation. a EGR1 mRNA expression was determined by real-time PCR. EGR1 mRNA was detected after treatment with 10 and 20 μg/mL LL-37 for 0–6 hr. b ASCs were incubated with 20 μg/mL LL-37 for 0–6 hr. Cell lysates were collected for western blot analysis. The EGR1 protein level was increased by LL-37 treatment in a time-dependent manner. c ASCs were treated with LL-37 and transfected with siRNA targeting EGR1 or a negative control sequence. After stabilization, cells were collected and lysed by the addition of 200 μL of cell lysis buffer. EGR1-targeting siRNA transfection was quantified by performing an EGR1-specific ELISA of cell lysates. d Cells that migrated after EGR1-targeting siRNA transfection were imaged by microscopy and counted using Scanscope. Results are expressed as the mean ± SD of three independent experiments. *, P < 0.01 vs. control; §, P < 0.01 vs. LL-37-treated cells transfected with negative control siRNA. e The effects on human ASC proliferation were similar to those on migration. A representative experiment from three independent experiments is shown. Bars represent the mean ± SD. *, P < 0.01 vs. control; §, P < 0.01 vs. LL-37-treated cells transfected with negative control siRNA. EGR1 early growth response 1, ASC adipose-derived stromal/stem cells, siRNA small interfering RNA, ELISA enzyme-linked immunosorbent assay
Mentions: We investigated the target genes of LL-37 that induce ASC proliferation and migration using the Agilent human 4x44K array, a human signaling pathway finder. Microarray analysis showed that LL-37 treatment significantly increased EGR1 expression by 18.47-fold (Table 1 and Additional file 3: Table S2). LL-37 treatment also increased the levels of several genes (including EGR2, early growth response 2; KLF10, Krupple-like factor 10; FOS; and CTGF, connective tissue growth factor) linked to various functions, such as the cell cycle, cell migration, cell proliferation, and transcription. To confirm the effect of LL-37 treatment on EGR1 expression in ASCs, cells were treated with 10 or 20 μg/mL LL-37 for different amounts of time and then real-time PCR analysis was performed. LL-37 treatment significantly increased the EGR1 mRNA level, which peaked at 1 hr and returned to the basal level at about 6 hr (Fig. 2a). Because LL-37 treatment considerably increased the mRNA level of EGR1, we attempted to ascertain whether it also increased the protein level of EGR1 via western blot analysis. LL-37 treatment rapidly increased the protein level of EGR1 in ASCs in a time-dependent manner (Fig. 2b). In addition to western blot analysis, ELISA analysis of cell lysates demonstrated that EGR1 production was significantly increased by LL-37 treatment (Additional file 2: Figure S1b)Table 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