LL-37 stimulates the functions of adipose-derived stromal/stem cells via early growth response 1 and the MAPK pathway.
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.
Department of Plastic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Yoolhee
Y
Yang
Department of Plastic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Hyunju
H
Choi
Department of Plastic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Mira
M
Seon
Department of Life Science, Sookmyung Women's University, Seoul, Korea. cdhkor@sookmyung.ac.kr.
Daeho
D
Cho
Department of Plastic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. si55.bang@samsung.com.|Bio-Med Translational Research Center, Samsung Medical Center, Seoul, Korea. si55.bang@samsung.com.
Sa Ik
SI
Bang
D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D000273:Adipose Tissue / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D000505:Alopecia / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism; D054377:Chemokine CXCL12 / Q000235:genetics / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D051766:Early Growth Response Protein 1 / Q000819:agonists / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D006197:Hair / Q000187:drug effects* / Q000254:growth & development; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013947:Thymosin / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics* / Q000378:metabolism; D054804:Cathelicidins; D000099201:Thymosin beta(4)
D023181:Antimicrobial Cationic Peptides; D018932:Chemokine CCL2; D054377:Chemokine CXCL12; D017077:Culture Media, Conditioned; D051766:Early Growth Response Protein 1; D048031:JNK Mitogen-Activated Protein Kinases; D012333:RNA, Messenger; D013947:Thymosin; D042461:Vascular Endothelial Growth Factor A; D048051:p38 Mitogen-Activated Protein Kinases; D054804:Cathelicidins; D000099201:Thymosin beta(4); C111835:CCL2 protein, human; C497325:EGR1 protein, human; C467484:VEGFA protein, human
Adipose-derived stromal/stem cells; Cell migration; Early growth response 1; Hair growth; LL-37; MAPK pathway; Mesenchymal stem cells; Paracrine actions; Proliferation; Regeneration