JNK/MAPK Signal Pathway Transduces ActivinB Signals To Regulate Skin Wound Healing

Skin wound healing is a complex biological process which includes inflammation, the renewed organization and reconstruction. One of the most important things during wound healing is that epithelial cells of the remnants around wound and the cells of subsidiary structure migrate and cover the wound. During the process of cell migration, there are a series of phenotypic changes; another thing is cell proliferation. Epidermal cells healing are the key to all layers of skin repair. Epidermal keratinocytes are the cells which have the ability of proliferation and differentiation. They play a key role in wound healing. Activative keratinocytes around the wound proliferate and migrate to complete re-epithelialization. Therefore, activation, proliferation, differentiation and migration of keratinocytes are the key to skin wound healing. Another one kind of cells which is involved in wound healing is fibroblasts. Wound contraction and granulation tissue formations after the traum are in favor of the migration of keratinocytes to cover the wound. Wound contraction is completed mainly by some fibroblasts which have the ability of contraction—myofibroblast. During the process of the formation of granulation tissue, fibroblasts also play an important role in (the production of collagen fibers). Activin (ACT) is considered to play an important regulatory role in tissue repair and re-epithelialization process. Our previous studies have found that In vitro a certain concentration of Activin B can accelerate wound healing by promoting the migration of keratinocytes through MEKK1/JNK signal pathway.1. JNK/MAPK signal pathway transduces ActivinB signals to regulate skin wound healing and hair follicle regeneration(1) Purpose In order to further study In vivo whether Activin B can promote the migration of keratinocytes to accelerate wound healing, and to explore the mechanism JNK/MAPK signal pathway and whether Activin B influence on initiation and development of hair follicle.(2) Methods Using the wound model, HE staining, oil red 0 staining and then using light microscope to observe the wound healing; the application of electron microscopy technique to observe the morphology and ultra structure of wound healing process; to observe the expression of K15, K19 by; immunohistochemistry; using Brdu to label and observe changes of cell proliferation during wound healing. The application of Western Blotting to detect the expression of P-JNK, P-ERK, P38 protein around wound healing.(3) Results1) Light microscope observation of the wound healing:In vivo, using mouse wound model, our research had shown that Activin B also can promote wound healing. Compared with the PBS group, the healing rate of Activin B group was significantly increased (P<0.05), while there was regeneration hair follicles in a base layer of epithelial tissue, and during late tissue reconstruction, the structure of the skin Activin B group are more integrity than the PBS group.2) The utilization of electron microscopy technique to observe the morphology and ultra structure of wound healing process:Through scanning electron microscope trauma surfaces, there were a large number of collagen and exudating cells in wound granulation tissue. There was the re-epithelialization skin at the junction of normal tissue and granulation and those surface keratinized are less than the surrounding normal squamous epithelium. The cells crawled along the collagen were also observed in Activin B group. In order to further study, we use scanning electron microscopy to observe cross-section wound. There are a large number of keratinocytes in the cross-section of Activin B group, while the number of keratinocytes in the PBS group cross-section wound was observably lower than one of Activin B group. Therefore, we believe that Activin B can promote proliferation and migration of keratinocytes In vivo. Using TEM to observe the morphology of basal keratinocytes, the results show that caudal cells of Activin B group are in dissociation state with the surrounding matrix, which makes for cell migration. On the contrary, caudal cells of PBS group are closely linked with the surrounding matrix. Therefore, we believe that In vivo Activin B can promote the migration of keratinocytes.3) The expression of K15, K19 and Brdu labeling cell proliferation:K15 expression was positive at the basal layer of normal skin around wounds at the fifth day after wound, and yet there were very little K15-positive cells near the thickened wound epithelium. There were very little K15-positive cells of PBS group in the thickened wound epithelium, but no K15-positive cells of Activin B group. So we think that Activin B through someone pathway inhibit the expression of K15, then promote proliferation and migration of keratinocytes. Different with decreased expression of K15, K19 expression increased early after surgery, and the K19 positive cells of Activin B group around wound were significantly increased compared with PBS, later the expression of K19 was reduced until to 13 days when re-epithelialization two groups had been completed, and there was little K19-positive cells in the bottom, but the area of one-third of the upper and lower parts of the outer root sheath of hair follicles had the K19-positive expression cells in Activin B group around wound. We further used Brdu to detect cell proliferation around the wound found that the number of Brdu-positive cells in epidermal basal layer and hair follicle of Activin B group was significantly higher than PBS group. Accordingly, we further believe that Activin B through someone signaling pathway regulate the biological activity of keratinocytes, thus promote proliferation and migration of keratinocytes.4) The results of Immunohistochemistry and Western Blotting:The phosphorylation activity around wound was detected by immunohistochemistry. The results were found:the expressions of p-JNK, p-cJun during 1st day to 6th day in Activin B group were positive, while the PBS group was negative. Different with p-JNK, p-cJun, p-ERK, p-P38 in the two groups were expressed in the epidermis. Further, using Western Blotting to detect the expressions of p-JNK, p-ERK, and p-P38 after traumatic injury, the results were found:Early from 3h to 12h after wound, p-JNK was expressed in two groups, but p-JNK in Activin B group was still expressed from 1st day to 5th day, while the expression of p-JNK in PBS group decreased. P-ERK and p-P38 in both groups were expressed during 3h-5th day. The results from immunohistochemistry and Western Blotting showed:Activin B may be passed through JNK, but not ERK, and P38, to regulate wound healing.(4) Conclusion Above results suggest that in vivo Activin B can promote keratinocytes proliferation and migration to promote healing, this signal may be mediated by the JNK/MAPK signal pathway.2. The effect on fibroblast activity of Activin B and the relevant signaling pathways(1) Purpose In vitro to investigate whether Activin B regulate the contractile function of fibroblast cell motility and signaling pathway.(2) Methods To isolation and culture of C57BL/6 mouse skin fibroblasts, and using Phallotoxins staining to observant the change of fibroblasts cytoskeleton proteins; Then to detect the phosphorylation activation of P-JNK, p-ERK, p-p38, P-cJun in fibroblast by Western Blotting.(3) Results1) Phallotoxins staining:Activin B stimulated fibroblasts,30min later F-actin turned from a small amount of concentrate in the cell membrane to distribution in the vicinity of the cytoplasm. This state had continued until 6h after stimulation. EGF stimulated fibroblast cells but lower F-actin was here after 1h. F-actin still concentrated in the cell membrane in PBS Group.2) The results of Western Blotting:There was the phosphorylation activation of p-JNK, p-ERK, p-P38, P-cJun protein after Activin B stimulated fibroblasts. Compared with Activin B group, phosphorylation activation of p-JNK, p-ERK, p-p38, p-cJun in the PBS group is not less. The phosphorylation activation of P-P38 is shown in Activin B stimulated fibroblasts 10min later, followed by slightly lower expression. The expression of p-cJun in fibroblasts was increased after Activin B stimulation 30min until 120min. After the application of the specific JNK inhibitor, the expression of p-JNK and p-c-Jun were negative. These results suggest that:p-JNK pathway transduces Activin B signals to regulate changes of fibroblast F-actin.(4) Conclusion These results suggest that:P-JNK, P-ERK, P-P38 pathway may be involved in fibroblast F-actin changes.3. The effects of Act B on the development of mouse hair follicle(1) Purpose To investigate the effect of Activin B on mouse hair follicle development and cycle.(2) Methods Using HE staining, alkaline phosphates staining (AP activity staining), oil red O staining, staining of apoptotic cells and tissue culture to observe the Activin B on the impact of neonatal hair follicle development and on adult mouse hair follicle cycle.(3) Results 1) Cultured neonatal rat back skin:compared with PBS group, the number of hair follicles of cultured neonatal rat back skin born 1 day in 5ng/ml Activin B group and 10ng/ml Activin B group were notedly higher, the differences were statistically significant (p= 0.01, p= 0.023), while the difference between the 5ng/ml Activin B group and 10ng/ml Activin B group was not statistically significant (p= 0.171). So we found that Activin B can induce the initiation of hair follicles.2) To smudge Activin B on rat back skin after birth:1-day neonatal birth, back skin was pink, and after born 3 days the color of suckling mice skin was light gray slightly and was no significant differences between PBS group and Activin B group. But Activin B group neonatal rat back skin was black, and there were the hairs after born 5th day. While the PBS Group neonatal back skin was gray. The number of hair follicles in Activin B at 3rd day after born was significantly higher than the number of hair follicles in PBS group (P= 0.00), and this further suggests Activin B can induce the initiation of hair follicles and promote the development of hair follicles.3) Using C57BL/6 mice to make depilation induced models:because hair follicles in telogen stage on rat back skin entered into anagen stage at the same time after depilation, we smudged Activin B on rat back skin after depilation. The results showed that compared with the PBS group, the color of mouse back skins of Activin B group first turned pink of telogen stage into black of anagen stage. Morphology observation further confirmed Activin B could speed up the hair follicles from the telogen stage to enter the anagen stage.(4) Conclusion The above experiments suggest that Activin B play an important role in initiation, development and cycle of hair follicles. This study provides a theoretical basis for the problem about hair follicle regeneration during wound healing.