MAPK Signal Pathway Transduces Activin A、Activin B Regulating Fibroblast

Chapter one MAPK signal pathway transduces Activin A, Activin B regulating fibroblastResearch backgroud and objective:Tissue repair and wound healing are two fundamental problems facing medical treatment currently. According to the definition in clinical medicine, wound healing is an intricate process in which the skin (or another organ-tissue) repairs itself after injury. It is a complex combination of regeneration of all sorts of organs and proliferation of granulation tissue, showing synergistic action of all processes. Skin wound healing is the most basic model of tissue repair of human organs and a hot topic in present-day scientific research.Skin is the soft outer covering of body. In human, the skin is the largest organ of the body made up of multiple layers, which are epidermis,Dermis and hypoderm,with functions of protecting the body, insulation, temperature regulation and sensation. The epidermis is composed of the outermost layers of the skin. The dermis is the layer of skin beneath the epidermis The dermis provides tensile strength and elasticity to the skin through an extracellular matrix composed of collagen fibrils, microfibrils, and elastic fibers. The hypodermis is not part of the skin, and lies below the dermis. It consists of loose connective tissue and elastin. Because it interfaces with the environment and is the first line of defense from external factors, it is susceptible to damage. In addition, skin wound caused by chronic diseases increase nowadays. As a result, Skin wound healing is an urgent problem to be addressed in clinical medicine. Skin wound healing is a complex biological process, kick starting a set of complex biochemical events, is divided into three sequential phases:inflammatory, proliferative and remodeling.Fibroblasts are the most common cells of connective tissue and major cells of Dermis of the skin in animals. At early stage of skin wound healing, fibroblasts secret extracellular matrix, including precollagen, fibronectin and collagen, etc. It makes up granulation tissue, together with new capillaries, inflammatory cells, and so on. It creates conditions for covering epidermic cells. Meanwhile, it can migrate to the wound to proliferate and promote wound healing. Fibroblasts which participate in wound healing derive from partially fibroblasts and undifferentiated mesenchymal cells in papillary layer of dermis, as well as fibroblasts and pericytes around blood vessel between. On the one hand the fibroblasts derive from cell proliferation and cell division, on the other hand, they come from neighboring mesenchymal cells, fibrocytes and capillary pericytes evoluting or migrating. At late stage of skin wound healing, fibroblasts secrete collagenase to reconstruct healing tissue. Fibroblasts play an important role in the formation of scar tissue which is characterized by fibrous connective tissue due to excessive synthesis and deposition of collagen. It is agreed that fibroblasts are the key cells in wound healing and scar formation.During wound healing cell growth factors participate and regulate each step of healing in addition to cell proliferation, migration and differentiation. The main cell growth factors have epidermal growth factor (EGF), fibroblast growth factor (bFGF), transforming growth factor beta(TGF-beta), platelet-derived growth factor (PDGF) and so on. Activin is a member of the transforming growth factor p (TGF-β) superfamily, has a wide range of biological activities, through its signaling transduced pathway regulating reproduction and embryonic development, modulating erythrocyte differentiation, participating in pathological inflammatory processes, inducing cell apoptosis, promoting wound healing after injury, and participating in the formation of organ fibrosis.Our team has found mice of ablating MEKK1 gene appear eyelid closure obstacles at birth, which is due to abnormal epidermal cell migration, JNK-MAPK and RhoA signaling pathway mediate Activin B regulating skin wound healing through regulation of the cytoskeleton and migration of keratinocytes. Mitogen-activated protein kinase (MAPK) is signaling system which is composed by cascade activation of serine/threonine protein kinase to mediate cell biological response, widely presenting in mammals, playing an important role in cell migration, differentiation, proliferation and other biological functions. Because fibroblasts play an important role in skin wound healing process, we hypothesize that MAPK may also mediate Activin regulation of fibroblasts. In order to explore MAPK whether or not to participate and how to mediate Activin regulation of fibroblasts, we used Activin to stimulate fibroblasts and observe fibroblasts change, and then blocked or activated particularly the signaling pathway to explore MAPK whether or not to be involved in Activin regulation of fibroblasts and its possible downstream signaling molecules, revealing molecular mechanisms of fibroblasts effect in the process which Activin regulated fibroblasts.We studied MAPK-mediated activin A, activin B regulation of fibroblasts in vitro to lay the foundation for studying how MAPK pathway to mediate Activin A, Activin B regulation of fibroblasts in cutaneous wound healing. The research aims to provide a new theory to the clinical treatments of skin wounds and scars.Research methods:Isolated and cultured C57BL/6 neonatal mice skin fibroblasts.The fibroblasts between fourth generation and sixth generation were divided into the Activin A group, Activin B group and PBS group, then we used Phallotoxins staining to observe fibroblasts cytoskeleton changing after stimulating 30min,1 hour, 2 hours,6 hours and 12 hours later. The dosage of Activin A application was 20ng/ml and Activin B was 10ng/ml.The fibroblasts between fourth generation and sixth generation were divided into Activin A 10 minutes group, Activin A 30 minutes group, Activin A 120 minutes group, Activin B 10 minutes group, Activin B 30 minutes group, Activin B 120 minutes group and PBS group, then we extracted fibroblasts proteins and used Western blot to detect phosphorylation activity of of p-JNK, p-ERK and p-p38.The fibroblasts between fourth generation and sixth generation were treated with the specific JNK inhibitor SP600125(5umol) or the p38 specific inhibitor SB202190(5umol) or ERK specific inhibitor SL327 (5umol) and the process keep 30 minutes. The fibroblasts were given Activin A or Activin B stimulation after the process. Then we use Phallotoxins staining to observe fibroblast cytoskeletal protein changes induced by Activin A and Activin B, and Western Blot was used to detect the expression of fibroblast p-JNK, p-ERK and p-p38.The fibroblasts between fourth generation and sixth generation were divided into the Activin A group, Activin B group and PBS group, then we scratched cells and observed cells migration after scratching 24 hours,48 hours and 72 hours later. Research result:The results of Phallotoxins Staining:F-actin changed from a little concentrating near the cell membrane little into a great quantity distributing throughout the cytoplasm. The change begain at 30 minutes after Activin A and Activin B stimulation and became most obvious at 2 hours after Activin A and Activin B stimulation, then the change induced by Activin A started to weaken and came back to the level induced by PBS at 12 hours after Activin A stimulation. The change induced by Activin B keep strong 6 hours after Activin B stimulation, and then the change begain to weaken and came back to the basic level. When fibroblasts were given specific ERK inhibitor(SL327),the aggregation of F-actin induced by Activin A and Activin B-induced fibroblast weakened, but not completely suppressed. When fibroblasts were given the specific JNK inhibitor(SP600125), Activin A, Activin B-induced fibroblast F-actin aggregation was completely inhibited. When fibroblasts were given specific p38 inhibitor(SB202190), Activin A and Activin B-induced fibroblast F-actin aggregation was not suppressed.The results of western blot:The p-JNK and p-ERK expression in Activin A and Activin B group were increased comparing to the PBS group, but p-p38 expression had no significant change. The p-JNK, p-ERK expression was high at 10 minutes after Activin A and Activin B stimulation and then reduced down to the pre-stimulus level. When JNK specific inhibitors were given, the p-JNK was inhibited, When ERK specific inhibitors were given, p-ERK was inhibited. With given a specific inhibitor of p38, p-p38 had no obvious changes. The results of western blot suggested JNK, ERK/MAPK mediate Activin A, Activin B regulating fibroblast cytoskeleton changes, but p38 was not found to be involved in fibroblast cytoskeleton changes induced by Activin A and Activin B.The results of cell scratching:There were no differences in fibroblast migration between the Activin A group, Activin B group and PBS group.Research conclusion:Activin A and Activin B regulate fibroblast cytoskeleton changes through JNK, ERK signaling pathway, the p38 pathway is not found to be involved in the regulation of fibroblast cytoskeleton induced by Activin A and Activin B.Chapter two Identified mice of epidermis-specific Cdc42, Rac 1 knocked out and construct relative plasmidResearch background and objective:Rho family is Guanylic acid triphosphatase and also known as G protein. The main members of the Rho family have Rho (RhoA, RhoB, RhoC), Rac (Racl, Rac2), Cdc42. Rho family proteins are important intracellular signaling molecules that regulate cell function through a variety of signaling pathways. Xunwei Wu and his co-workers found Cdc42 and Racl knockout transgenic mice had their hair growth and development problems and abnormal hair follicle. The hair follicles are the basis to maintain hair growth, impact skin wound healing through a variety of ways.Hair follicle stem cells are locating in the hair follicles, which are believed to be one kind of seed cells of the skin wound healing by many scholars. Meanwhile regeneration of skin subsidiary organ such as the hair follicle is an important standard to measure the healing quality. It is reported that in vitro the Rho-GTPase family may modulate cell migration, proliferation, and differentiation of a variety of biological functions through the regulation of cell adhesion plaques, stress fibers, filopodia and lamellipodia, which is involved in skin wound healing. Therefore, such studies show Rho-GTPase in the skin wound healing play an important regulatory.To study the role of Rho-GTPase in skin wound healing, we generated the epidermis-specific Cdc42, Rac1 gene knockout mice and then constructed lentiviruses of Cdc42 dominant negative effect mutant (Cdc42NA) and constitutively active mutant (Cdc42L61) for the future to explore the Cdc42 protein’s role in the skin wounds healing and its upstream and downstream signaling pathway.Research methods:The mice of type KRT14-Cre, Racl-loxP, Cdc42-loxP were purchased or given as gifts. To generate mice with restricted deletion of the Racl or Cdc42 gene, we hybridized KRT14-Cre mice with Racl-loxP or Cdc42-loxP mice. We used Isopropanol or DNA extraction kit to extract mice DNA. UV spectrophotometer was used to measure DNA concentration and purity, then measurement data was recorded. We analyzed date using two independent samples t-test we thought P<0.05 is statistically significant. They were performed on SPSS statistical software. PCR primers were designed according to the DNA sequence of mice gene knocked out before and after, then PCR method was used to identify the mice genotypes.We construct the plasmid Plenti6/v5-Cdc42NA Plenti6/v5-Cdc42L61 by restrictive endonucleases and DNA ligase, then send recombinant plasmid to Invitrogen company to detect sequence. We used E.coli to amplify plasmid, then extracted and purified plasmid. The plasmid was identified by comparing the bands which were from the plasmid digested by restrictive endonucleases. We used ViraPower lentivirus producing system to make the corresponding lentiviruses. We detected EGFP fluorescence to estimate lentivirus prepared effect by 293FT cells.Research result:Cdc42-deficient homozygote mice died in 24 hours after birth, the heterozygote could live normally like the wild type mice. Racl-deficient homozygote mice lost nearly all of their hair within a few weeks after birth, the heterozygote could live normally like the wild type mice,it was consistent with report. DNA of isopropanol extraction had high purity and high concentration, and there was no significant difference in the concentrations of two groups. There was significant difference in purity, and DNA of isopropanol extraction had higher purity. The result of PCR identification showed the genes were knocked out successfully, and there were bands of genes knocked out.We used agarose gel electrophoresis to distinguish the segments which were from plasmid digested by restrictive endonucleases, the result suggested we constructed plasmid correctly. The sequence of plasmid is consistent with the sequence published in NCBI. We found green fluorescent coming from the 293 FT cells which produced lentiviruses by fluorescence microscopy, suggesting the successful preparation of the lentiviruses.Research conclusion:We generated mice with restricted deletion of the Racl or Cdc42 gene successfully. PCR identification method was stable and well. The method of isopropanol extraction was simple, effective, and had high practical value.We constructed the recombinant plasmid Plenti6/v5-Cdc42NA and Plenti6/v5-Cdc42L61 of Rho family correctly, then we produced lentiviruses of Cdc42 dominant negative effect the mutant (Cdc42NA) and constitutively active mutant (Cdc42L61) successfully. It is prepared for next experiments.