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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

Involvement of the MAPK pathway in LL-37-induced ASC proliferation and migration. a Human ASCs were treated with 20 μg/mL LL-37 for 1, 2, 4, 24, and 48 hr. After cell lysis, the levels of phosphorylated ERK1/2, p38, and JNK were determined by western blot analysis. The levels of total ERK1/2, p38, and JNK were used to confirm equal loading of the cell lysates. LL-37 treatment significantly increased the phosphorylation of ERK1/2, p38, and JNK. b ASCs were pretreated with or without a specific inhibitor of ERK1/2 (PD98059), p38 (SB203580), or JNK (SP600125) for 1 hr and then treated with 20 μg/mL LL-37. EGR1 protein levels in cell lysates were analyzed by an EGR1-specific ELISA. c LL-37-enhanced migration decreased in human ASCs treated with PD98059, SB203580, or SP600125. d LL-37-enhanced ASC proliferation was inhibited by treatment with specific inhibitors of ERK1/2, p38, or JNK (similar to ASC 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 ASCs. MAPK mitogen-activated protein kinase, ASCs adipose-derived stromal/stem cells, EGR1 early growth response-1
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Fig3: Involvement of the MAPK pathway in LL-37-induced ASC proliferation and migration. a Human ASCs were treated with 20 μg/mL LL-37 for 1, 2, 4, 24, and 48 hr. After cell lysis, the levels of phosphorylated ERK1/2, p38, and JNK were determined by western blot analysis. The levels of total ERK1/2, p38, and JNK were used to confirm equal loading of the cell lysates. LL-37 treatment significantly increased the phosphorylation of ERK1/2, p38, and JNK. b ASCs were pretreated with or without a specific inhibitor of ERK1/2 (PD98059), p38 (SB203580), or JNK (SP600125) for 1 hr and then treated with 20 μg/mL LL-37. EGR1 protein levels in cell lysates were analyzed by an EGR1-specific ELISA. c LL-37-enhanced migration decreased in human ASCs treated with PD98059, SB203580, or SP600125. d LL-37-enhanced ASC proliferation was inhibited by treatment with specific inhibitors of ERK1/2, p38, or JNK (similar to ASC 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 ASCs. MAPK mitogen-activated protein kinase, ASCs adipose-derived stromal/stem cells, EGR1 early growth response-1

Mentions: The MAPK pathway plays an important role in the migration and proliferation of MSCs and cancer cells [10, 28]. To elucidate the signaling mechanism involved in LL-37-induced ASC proliferation and migration in detail, we examined the effect of LL-37 on the MAPK pathway in human ASCs. Treatment with LL-37 significantly increased the levels of phosphorylated extracellular signal-regulated kinase (ERK) 1/2, p38, and c-Jun N-terminal kinase (JNK), but did not change the total levels of these proteins (Fig. 3a). To further determine the involvement of kinase phosphorylation in the increased migration, proliferation, and EGR1 production of ASCs, cells were treated with LL-37 in the presence and absence of a specific inhibitor of ERK (PD98059), p38 (SB203580), or JNK (SP600125). All these MAPK inhibitors (PD98059, SB203580, and SP600125) significantly reduced LL-37-induced EGR1 production (Fig. 3b). In addition, each of the inhibitors attenuated the LL-37-induced increase in ASC migration and proliferation (Fig. 3c and d). Taken together, these data suggest that: 1) LL-37 induces multiple signaling pathways, including those linked with ERK, p38, and JNK phosphorylation; and 2) these kinases are involved in the regulation of human ASC migration and proliferation. These results suggest that LL-37 stimulates ASC function through a MAPK-dependent mechanism.Fig. 3


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)

Involvement of the MAPK pathway in LL-37-induced ASC proliferation and migration. a Human ASCs were treated with 20 μg/mL LL-37 for 1, 2, 4, 24, and 48 hr. After cell lysis, the levels of phosphorylated ERK1/2, p38, and JNK were determined by western blot analysis. The levels of total ERK1/2, p38, and JNK were used to confirm equal loading of the cell lysates. LL-37 treatment significantly increased the phosphorylation of ERK1/2, p38, and JNK. b ASCs were pretreated with or without a specific inhibitor of ERK1/2 (PD98059), p38 (SB203580), or JNK (SP600125) for 1 hr and then treated with 20 μg/mL LL-37. EGR1 protein levels in cell lysates were analyzed by an EGR1-specific ELISA. c LL-37-enhanced migration decreased in human ASCs treated with PD98059, SB203580, or SP600125. d LL-37-enhanced ASC proliferation was inhibited by treatment with specific inhibitors of ERK1/2, p38, or JNK (similar to ASC 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 ASCs. MAPK mitogen-activated protein kinase, ASCs adipose-derived stromal/stem cells, EGR1 early growth response-1
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Related In: Results  -  Collection

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Fig3: Involvement of the MAPK pathway in LL-37-induced ASC proliferation and migration. a Human ASCs were treated with 20 μg/mL LL-37 for 1, 2, 4, 24, and 48 hr. After cell lysis, the levels of phosphorylated ERK1/2, p38, and JNK were determined by western blot analysis. The levels of total ERK1/2, p38, and JNK were used to confirm equal loading of the cell lysates. LL-37 treatment significantly increased the phosphorylation of ERK1/2, p38, and JNK. b ASCs were pretreated with or without a specific inhibitor of ERK1/2 (PD98059), p38 (SB203580), or JNK (SP600125) for 1 hr and then treated with 20 μg/mL LL-37. EGR1 protein levels in cell lysates were analyzed by an EGR1-specific ELISA. c LL-37-enhanced migration decreased in human ASCs treated with PD98059, SB203580, or SP600125. d LL-37-enhanced ASC proliferation was inhibited by treatment with specific inhibitors of ERK1/2, p38, or JNK (similar to ASC 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 ASCs. MAPK mitogen-activated protein kinase, ASCs adipose-derived stromal/stem cells, EGR1 early growth response-1
Mentions: The MAPK pathway plays an important role in the migration and proliferation of MSCs and cancer cells [10, 28]. To elucidate the signaling mechanism involved in LL-37-induced ASC proliferation and migration in detail, we examined the effect of LL-37 on the MAPK pathway in human ASCs. Treatment with LL-37 significantly increased the levels of phosphorylated extracellular signal-regulated kinase (ERK) 1/2, p38, and c-Jun N-terminal kinase (JNK), but did not change the total levels of these proteins (Fig. 3a). To further determine the involvement of kinase phosphorylation in the increased migration, proliferation, and EGR1 production of ASCs, cells were treated with LL-37 in the presence and absence of a specific inhibitor of ERK (PD98059), p38 (SB203580), or JNK (SP600125). All these MAPK inhibitors (PD98059, SB203580, and SP600125) significantly reduced LL-37-induced EGR1 production (Fig. 3b). In addition, each of the inhibitors attenuated the LL-37-induced increase in ASC migration and proliferation (Fig. 3c and d). Taken together, these data suggest that: 1) LL-37 induces multiple signaling pathways, including those linked with ERK, p38, and JNK phosphorylation; and 2) these kinases are involved in the regulation of human ASC migration and proliferation. These results suggest that LL-37 stimulates ASC function through a MAPK-dependent mechanism.Fig. 3

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