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Activating PKC-ζ Is Required for Migration of Human Epidermal Stem Cells Promoted by SDF-1

Author: GuoRui
Tutor: LiShiRong
School: Third Military Medical University
Course: Surgery
Keywords: PKC-ζ human epidermal stem cells stromal-derived factor 1 chemotaxis
CLC: R329
Type: Master's thesis
Year: 2011
Downloads: 68
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Abstract

Various trauma and war wounds make the body injured and organ impairment, cause the internal environment unbalance, infection, hemorrhage, pain and so on. Even shock to death. The skin can effectively guard against the harmful substances invasion, keep homeostasis, plays an important role in maintaining the body normal functions. When trauma and war wounds damage the body’s surface, Covering the wound and speeding up the wound healing as soon as possible help body avoid dehydration, injure and further infection. Skin injury healing generally divided into three stages:1 inflammatory reaction. 2 cell migration, proliferation and granulation. 3 new tissue reconstruction. It is a complex process involves in multiple cells, cells factor and extracellular matrix. How to accelerate wound healing is one of the hot areas where trauma and war wounds studies are being performed.People gradually realize the epidermal stem cells plays an important role In wound healing. The epidermal stem cells are located in the basal skin epidermis accounted for 1% ~ 10%. In addition to their self-renewing, high proliferation and multipotency, these cells are quiescent with a low tendency to divide. They are thought to be the important component in skin development and repair, contribute to the skin metabolism. The epithelization of the wounding involves activation, migration and proliferation of epidermal stem cells. The key about wound recovering is to enforce these tissue-specific stem cells function.The stem cells migrate to target from original position regulated by chemotactic factors/chemotactic factors receptors. Stromal cell derived factor-1 was discovered in culture supernatant fluid of bone marrow stromal cells. Stromal cell derived factor-1 and its specific receptor CXCR4 make up the SDF-1/ CXCR4 axis. It widely distributed in various tissues. It have been shown to join in the regulation of embryonic hematopoietic migration, embryonic development, hematopoietic generation, tissue remodeling, tumor cell transfer and wound healing. In the central nervous system development process, SDF-1 plays an equally important role in the migration of embryos nerve cells. Our previous study reported that CXCR4 is expressed at epidermal cells, The nearer to basal layer the higher expression were showed an the epidermal cells. The levers of SDF-1 in acute traumatic wound or the rim of the wound is more than the chronic wound. The increase of SDF-1 expression appears after skin wound. Studies of the dermal equivalent indicate that expression ofβ-1 integrin cells increase and migrate to the superbasal layer when the levers of SDF-1 increasing at wound. The injury of skin affect SDF-1-induced chemotaxis.The regulation mechanisms of SDF-1-induced the epidermal stem cell chemotaxis remain unclear.A prototype of the protein kinase C family of serine/threonine kinase was first described by Nishizaka in 1977. The PKC family is divided into three subgroups: classical PKCs (α、β1、β2、γ),novel PKCs (δ、ε、η、θ),and atypical PKCs(ι/λ、ζ).Classical cPKCs are calcium (Ca2+) sensitive and need in addition DAG or phorbol esters for activation. The novel nPKCs are calcium (Ca2+) independent but still require DAG or phorbol ester. For the atypical aPKCs, being also Ca2+ independent. Various PKC isoforms play different roles in cell signaling including apoptosis, angiogenesis, differentiation, proliferation, and migration. PKC isoforms require serine/threonine phosphorylation for their own activation.The PKC isoforms can be divided into three major groups based on their structual characteristics and cofactor requirements. PKC-αenhance the migration of capillary endothelial cells, it can also contribute to migratory activity of colon carcinoma cells and breast carcinoma cells.Other isoforms such as : PKC-β, PKC-δ, PKC-θ, PKC-ζbind to the migration of cells. Atypical PKC-ζregulates SDF-1-mediated migration and development of human CD34+ progenitor cells and mesenchymal stromal cells. Silencing PKC-ζexpression impaired migration of mouse peritoneal macrophages. At present, the regulation mechanisms of SDF-1-induced the epidermal stem cell chemotaxis remain unclear. For this reason, we isolated human epidermal stem cells from preputial skin samples, and utilized SDF-1 to promote the directional migration of ESCs, and explored the phosphorylation of signaling protein PKC-ζrelated mechanisms.Objection: To investigate the role of the phosphorylation of signaling protein PKC-ζin SDF-1-induced the epidermal stem cell chemotaxis in wound healing.Methods: 1.We isolated epidermal stem cells by adhering to collagen type IV after digesting the normal preputial skin samples and cultured in serum-free medium (DK- SFM).β-1 integrin and cytokeratin-19 are used to identify epidermal stem cells by immunohistochemical staining.2.Different PKC isoforms inhabitors (staurosporine, chelerythrine, PS-ζ) regulates SDF-1-induced the epidermal stem cell chemotaxis in transwell assay.3.Western blot analysis was used to analyze the phosphorylation of signaling protein PKC-ζupon SDF-1 stimulation at different time phase.4.Polarization and membranal protrusions are classical hallmarks of motile cells. Cell morphology and polymerized actin distribution were examined by phallodin staining.Results:1.Immunohistochemical staining revealed that the cells we cultured were positive forβ-1 integrin and cytokeratin-19.2.PS-ζgroup significantly reduced the extent of the epidermal stem migration.(P<0.05)3.Western blot analysis results suggest that PKC-ζmay play an important role in epidermal stem cell respone to SDF-1 stimulation.4.SDF-1 induced polarization was accompanied by membranal protrusions in epidermal stem cells. Supression of signaling protein PKC-ζdecrease SDF-1 induced polarization and actin polymerization.Conclusion:1.We can select and culture the epidermal stem cell in vitro.2.Along with the different PKC isoforms inhabitors used in transwell assay, results suggest that PS-ζgroup significantly reduced the extent of the epidermal stem migration.3.SDF-1 induce PKC-ζactivation.4.Supression of signaling protein PKC-ζdecrease SDF-1 induced polarization and actin polymerization.

Activating PKC-ζ Is Required for Migration of Human Epidermal Stem Cells Promoted by SDF-1

Abstract

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