Regulation Of IGF1 And TIMP1 On The Related Signal Pathways During Development Of Arbas Cashmere Goat Dermal Papilla Cells

As a kind of dermal cells of hair follicles,dermal papilla cells locate in the basal part of hair follicles,and play an important role in the development and regeneration of hair follicles.There are two kinds of hair follicles in the skin of cashmere goats,primary and secondary hair follicles,in which the dermal papilla cells correspond to primary and secondary dermal papilla cells.Wool growth is controlled by primary dermal papilla cells,and cashmere growth is controlled by secondary dermal papilla cells.Hair growth and hair follicle development is a complex regulatory process,which is the results of multiple signaling pathways regulation.It was found that the transition of hair follicles from telogen to anagen could be accelerated to promote hair development and growth when PI3K/AKT and ERK1/2 signaling pathways were activated.IGF1 and TIMP1 can directly and indirectly regulate PI3K/AKT and ERK1/2 signaling pathways.In this study IGF1 and TIMP1 genes were selected to explore their effects on AKT and ERK1/2 signaling pathways in primary and secondary dermal papilla cells,so that to provide experimental basis for further study of cashmere goat primary and secondary dermal papilla cells on hair follicle development and hair regeneration.1.Expression of IGF1,TIMP1,AKT and ERK1/2 signaling pathways related genes in dermal papilla cellsIn this study,in vitro growth status of primary and secondary dermal papilla cells was observed,and the expression of mRNA of IGF1,TIMP1 and the related genes of AKT and ERK1/2 signaling pathways of the cells was detected by real time-PCR.The results showed that mRNA expression levels of IGF 1,TIMP1,MMP9 and PTEN gene in primary dermal papilla cells werel8,2.1,9,1.9 times of that in secondary dermal papilla cells respectively,and the mRNA expression levels of VEGF and KDR in primary hair papilla cells were 0.71,0.08 times of that in secondary dermal papilla cells respectively.Expression of IGF 1,TIMP1,AKT and ERK1/2 protein and phosphorylation of AKT and ERK1/2 in primary and secondary dermal papilla cells were detected by Western Blot.The results showed that the protein expression of IGF 1 and TIMP1 in primary hair papilla cells were 2 and 1.9 times of that in secondary dermal papilla cells respectively,the protein expression of ERK1/2 and AKT in primary dermal papilla cells were 1.01 and 1.02 times of that in secondary dermal papilla cells respectively.The protein phosphorylation level of ERK1/2 in primary dermal papilla cells was 0.4 times of that in secondary dermal papilla cells,and the phosphorylation level of AKT in primary dermal papilla cells was 4.6 times of that in secondary dermal papilla cells.2.Vector construction and transfection analysisThe CDS sequences of IGF1 and TIMP1 were cloned by ordinary PCR.Overexpression vectors,pIRES2-EGFP-IGF1 and pIRES2-EGFP-TIMP1,were constructed with pIRES2-EGFP as skeleton vector.Five interference vectors,IGF1-14,IGF1-428,TIMP1-126,TIMP1-173 and TIMP1-510,were constructed as well.The overexpression and interference vector was transfected into the primary dermal papilla cells by electroporation,and the overexpression and interference efficiency of IGF 1 and TIMP1 was detected by Real time-PCR.The results showed that the overexpression efficiency of IGF 1 and TIMP1 was 4.8 and 2 times of control respectively.The best interference efficiency was obtained by IGF 1-14 and TIMP1-173,the interference efficiency was 0.54 and 0.39 times of control respectively.3.Effects of IGF1 and TIMP1 on the expression of related genes of AKT and ERK1/2 signaling pathwaysThe over expression vectors,pIRES2-EGFP-IGF1 and pIRES2-EGFP-TIMP1,and the interference vectors,IGF1-14 and TIMP1-173,were transfected into primary and secondary dermal papilla cells respectively,then cell samples were collected after 48 hours to extract RNA and protein.Transcription levels of IGF1,TIMP1 and AKT and ERK1/2 signal pathway related genes were detected by real-time quantitative PCR,and protein expression and protein phosphorylation level of IGF1,TIMP1,AKT and ERK1/2 were detected by Western blot.The results showed:3.1 Transcription levels of IGF1 and related genes in the dermal papilla cells:Transcription levels of AKT and ERK1/2 in IGF1 overexpressed primary dermal papilla cells were increased,and were 1.35 and 1.47 times of control respectively.Transcription level of VEGF was decreased in 0.43 times of the control.In IGF1 interference primary dermal papilla cells,transcription levels of AKT,ERK1/2 and VEGF were decreased,and were 0.60,0.71 and 0.56 times of control respectively.The AKT,ERK1/2 and VEGF transcription levels in IGF1 overexpressed secondary dermal papilla cells were decreased,were 0.73,0.67 and 0.40 times of the control respectively.The AKT and ERX1/2 transcription levels in IGF1 interference secondary dermal papilla cells were higher than that of control,were 1.45 and 1.51 times respectively.The transcription level of VEGF was 0.84 times of control.The results showed that IGF1 promoted transcription of AKT and ERK1/2 in primary dermal papilla cells,whereas in secondary dermal papilla cells,IGF1 inhibited transcription of AKT and ERK1/2.In primary and secondary dermal papilla cells,overexpression and interference of IGF1 inhibited transcription of VEGF.3.2 Protein phosphorylation levels of IGF 1 and related genes in dermal papilla cells:Phosphorylation levels of AKT and ERK1/2 were 1.72 and 1.61 times of the control respectively in IGF1 overexpressed primary dermal papilla cells,while they were 2.10 and 1.52 times of the control respectively in IGF1 interference primary dermal papilla cells.Phosphorylation levels of AKT and ERK1/2 were respectively 4.23 and 0.62 times of the control in IGF1 overexpressed secondary dermal papilla cells,while they were 5.68 and 0.78 times of the control respectively in IGF1 interference secondary dermal papilla cells.The results showed that overexpression and interference of IGF1 upregulated the phosphorylation of AKT in primary and secondary dermal papilla cells.Overexpression and interference of IGF1 increased the phosphorylation level of ERK1/2 in primary dermal papilla cells,whereas overexpression and interference of IGF 1 inhibited the phosphorylation of ERK1/2 in secondary dermal papilla cells.3.3 Transcription levels of TIMP1 and related genes in dermal papilla cells:The VEGF,KDR,MMP9 and PTEN transcription levels were decreased in TIMP1 overexpressed primary dermal papilla cells,which were 0.73,0.65,0.58 and 0.45 times of control respectively.The VEGF,KDR,MMP9 and PTEN transcription levels were increased in TIMP1 interference primary dermal papilla cells,which were 1.26,1.54,1.09 and 1.05 times of the control respectively.The transcription levels of VEGF,KDR,MMP9 and PTEN were decreased in TIMP1 overexpressed secondary dermal papilla cells,which were 0.33,0.21,0.28,and 0.47 times of the control group respectively.The transcription levels of VEGF,KDR,MMP9 and PTEN were increased in TIMP1 interference secondary dermal papilla cells,which were 1.05,1.35,1.38 and 1.95 times of control group respectively.The results showed that TIMP1 inhibited the transcription of MMP9,VEGF,KDR and PTEN in primary and secondary dermal papilla cells.3.4 Protein phosphorylation levels of TIMP1 and related genes in dermal papilla cells:Phosphorylation levels of AKT and ERK1/2 in TIMP1 overexpressed primary dermal papilla cells were 0.6 and 2.1 times of control,and they were 1.5 and 6.4 times of control in TEMP1 interference primary dermal papilla cells,respectively.Phosphorylation levels of AKT and ERK1/2in TIMP1 overexpressed secondary dermal papilla cells were 0.7 and 0.63 times of control,they were 1.4 and 0.9 times of control in TIMP1 interference secondary dermal papilla cells,respectively.The results showed that TIM1 inhibited the protein phosphorylation level of AKT in the primary and secondary dermal papilla cells,in primary dermal papilla cells,overexpression and interference of TIMP1 promoted phosphorylation of ERKl/2,overexpression and interference of TIMP1 inhibited the phosphorylation of ERK1/2 in secondary dermal papilla cells.In conclusion:ERK1/2 signaling pathway could be activated by overexpression and interferes of IGF 1 and TIMP1 in primary dermal papilla cells.ERK1/2 signaling pathway could be inhibited by overexpression and interferes of IGF 1 and TIMP1 in secondary dermal papilla cells.AKT signaling pathway could be activated by overexpression and interferes of IGF 1,and could be inhibited by TIMP1 overexpression in primary and secondary dermal papilla cells.