| Beef is considered as one of the healthy foods for human because of its abundant nutrient. Its flavor and tenderness can be improved by inducing intramuscular fat composition. Previous studies in vitro and in vivo on various kinds of animals elucidated that adipocytes proliferation and differentiation at cellular level were regulated by some important transcription factors. CCAAT-enhancer-binding protein family (C/EBPs) as one of these transcription factor families played pivotal role in the control of fat formation as well as fatty acids metabolism. However, there is little information concerning the function of bovine C/EBPα, C/EBPβ and C/EBPδ genes. Herein, we detected the potential specific effects of the three genes and their possible interactions during bovine fibroblaststrans-differentiation process. Furthermore, we also investigated their effects on several other fat cell differentiation related genes. The main results were as follows:(1) The complete CDS sequences of bovine C/EBPα, C/EBPβ and C/EBPδ genes were successfully obtained. The CDS areas of bovine C/EBPα, C/EBPβ and C/EBPδ genes were1062bp,1047bp and771bp in length, coding353,348,256amino acid residues respectively. The putative amino acid sequences showed bovine C/EBPα, C/EBPβ and C/EBPδ shared higher similarities of95%,97%and87%with their homologue respectively. Bovine C/EBPα and C/EBPβ occupied one outside to inside transmembrane domain, locating within amino acid from233to252and from190to208respectively, while C/EBPδ occupied one inside to outside transmembrane domain, locating within amino acid from125to141. Besides, all the three putative proteins had one Basic leucine zipper domain (bZIP) consisted of a helixes, consistent with the predominant characteristic of C/EBPs.(2) Recombinant adenovirus carrying bovine C/EBPα, C/EBPβ and C/EBPδ complete CDS fragments were successfully constructed, and further packaged and amplified in HEK293A cell lines.(3) Oil O staining results demonstrated that over-expression of C/EBPα, C/EBPβ or C/EBPδ converted bovine fibroblasts into adipocytes or adipocytes-like cells. Over-expression of the combination of the two or the three isoforms converted fibroblasts into fat-laden adipocytes. (4) Real-time PCR results indicated over-expression of C/EBPα or C/EBPβ enhanced the mRNA expression of each other during bovine fibroblasts trans-differentiation, while none of them affected C/EBPδ mRNA expression. Meanwhile, simultaneous up-regulation of C/EBPβ and C/EBP5enhanced C/EBPa mRNA expression at some level.(5) Real-time PCR results indicated over-expression of C/EBPα and/or C/EBPβ upregulated PPARy mRNA expression level in the late phase of cell differentiation, while C/EBP8did not. All the three genes had no significant influence on SREBP1gene.(6) Real-time PCR results indicated over-expression C/EBPα or C/EBPβ upregulated the mRNA expression level of genes related to adipocytes such as Lipoprotein lipase (LPL), Acetyl-CoA carboxylase alpha (ACC-a), Fatty acid synthase (FAS), Stearoyl-CoA desaturase (SCD), Fatty acid transporter member1(FATP1) and Complement Factor D (Adipsin/CFD) genes. Over-expression of C/EBPδ increased the mRNA expression level of LPL, CFD and Fatty acid binding protein4(FABP4) genes. Combination of C/EBPα, C/EBPβ and C/EBPδ over-expression enhanced the expression of the above mentioned genes except FABP4and FATP1.(7) The mRNA level of FABP4gene significantly increased only in cells in which C/EBP8was upregulated, on the contrary, it decreased when C/EBPβ was upregulated to some extent, implying the two C/EBP isoforms may have dissimilar effects on FABP4gene at cellular level in bovine.In conclusion, we successfully converted bovine fibroblasts into adipocytes or adipocytes-like cells by over-expressing bovine transcription factors namely C/EBPα, C/EBPβ and C/EBPδ, either individually or by combination. Also a simple sketch map between the three transcription factors interaction, and their effects on other related genes was obtained based on the present results. |
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