Molecular Cloning, Expression Profiles And Regulation Analysis Of Insulin-like Growth Factor Binding Protein(IGFBPs) In Japanese Flounder (Paralichthys Olivaceus)

The insulin-like growth factor (IGF) system plays a crucial role in stimulatingcell growth, metabolism and reproduction in fish and other vertebrates. It is comprisedof the ligands (IGF-I and IGF-II), cell surface receptors (IGF-IR and IGF-IIR), andbinding proteins (IGFBP1-6). In addition to the functions of modulating IGFsavailability, stimulating or inhibiting the biological activities of IGFs, IGFBPs alsohave IGF-independent actions for cell growth or differentiation via their putativereceptors on cell membrane or direct nuclear actions by transporting into the nucleus.In this study, we cloned and characterized cDNA sequence of insulin-like growthfactor binding protein-2a,-2b,-3and-4(IGFBP-2a,-2b,-3and-4) from Japaneseflounder (Paralichthys olivaceus). Quantitative real-time PCR (qPCR) was performedto analyze the tissue distribution and temporal expression of IGFBPs mRNA duringembryonic development as well as larvae and juveniles. The effects of hormonalregulation on IGFBPs expression were investigated using the primary culturedhepatocytes of Japanese flounder. Furthermore，the promoter sequences of Japaneseflounder IGFBPs genes were obtained and several potential transcription factor (TF)binding sites were determined. The main topics of this paper are as followed：（1） We obtained two IGFBP-2genes from Japanese flounder. IGFBP-2a1186bplength with a42bp5’UTR, a283bp3’UTR and a861bp ORF which encoded apro-peptide of286amino acid residues, including a putative signal peptide of26residues; IGFBP-2b cDNA sequence was1075bp in length, containing complete ORFwith819bp which encoded a pro-peptide of248amino acid residues and had aputative signal peptide of24residues. Two typical IGFBP motif (GCGCCXXC),18 cysteine residues and Arg-Gly-Asp (RGD) motif were presented in the Japaneseflounder IGFBP-2s. Phylogenetic analysis demonstrated that Japanese flounderIGFBP-2a and-2b clustered within the IGFBP-2group, suggested that they originatedfrom one ancestral gene and generated because fish genome duplication. Japaneseflounder IGFBP-2a and-2b were predominantly distributed in liver. During the earlyembryonic stage, the expression level of IGFBP-2a was obvious higher thanIGFBP-2b. However, IGFBP-2b expression gradually increased and exceeded theexpression level of IGFBP-2a after hatching. These all result suggested that IGFBP-2awas mainly involved in the early embryonic development, and IGFBP-2b may playprimary roles in regulating growth and development of larvae and juveniles.（2） The full-length cDNA sequence of Japanese flounder IGFBP-3was1060bp,including a5’ UTR of102bp, a3’UTR of97bp and an ORF of861bp encoding aprotein of286amino acids. A putative signal peptide of22residues was predicted.18cysteine residues, two conserved IGFBPs motif (GCGCCXXC and CWCV), nuclearlocalization sequence (NLS), heparin binding domain(HBD) and metal bindingdomain(MBD) were found in the N-and C-terminal domain. The genomic sequenceJapanese flounder IGFBP-3was15223bp including4exons and3introns. Alignmentand phylogenetic analysis indicated that Japanese flounder IGFBP-3was the memberof fish IGFBP-3family. qPCR analysis demonstrated that IGFBP-3mRNA wasdetected in all selected tissues, with the most abundant in ovary. Maternal transcriptsof IGFBP-3gene existed in the early embryonic stages. Expression level of IGFBP-3mRNA was relatively lower at7dph, and significantly increased after themetamorphosis period. All these results demonstrated that IGFBP-3may be involvedin the fish early embryonic development.（3） The full-length cDNA of Japanese flounder IGFBP-4was1493bp, containingan open reading frame (ORF) of780bp, a5’-untranslated region (UTR) of552bp ofand a3’UTR of161bp. The complete ORF encoded a protein of259amino acids witha putative signal peptide of28residues. Japanese flounder IGFBP-4contained20cysteine residues and two conserved IGFBPs motif (GCGCCXXC and CWCV) in theN-terminal domain and C-terminal domain.13414bp genomic sequence of Japanese flounder IGFBP-4was obtained by genome walking and4exons and3introns wereidentified. Alignment and phylogenetic analysis revealed that it was indeed theortholog of the mammalian IGFBP-4gene. IGFBP-4was expressed in all selectedtissues from six healthy adults and was most abundant in intestine. It were detectedwith various level in unfertilized eggs and embryos at early stages, as well as larvaeand juveniles. After hatching, expression level of IGFBP-4mRNA was significantincreased in3days post hatching (dph), and gradually decreased during themetamorphosis period. The result suggested that Japanese flounder IGFBP-4mayplay important roles in regulating growth and development and be ontrolled bynutritional status. In addition, the mature peptide of Japanese flounder IGFBP-4wasexpressed in the form of fusion protein using pET-32a（+）as expression vectors andBL21（DE3）pLysS as expression host. SDS-PAGE analysis showed the most suitableexpression condition was continuous cultivation at20℃for6h.（4） In the primary cultured hepatocytes of Japanese flounder,10nM and100nMGH increased IGFBP-4mRNA levels. After6h treatment with10nM GH, the amountof IGFBP-4transcripts was rose and remained relatively higher level. In comparisonwith untreated cells, insulin reduced IGFBP-4mRNA levels in treated hepatocytes.Then the expression level of IGFBP-4continuously declined after6-48h treatmentwith10μM insulin. Different concentrations of IGF-I and IGF-II influenced IGFBP-4expression in a fluctuating manner. The level of IGFBP-4mRNA in treated cells wasobviously down-regulation with10ng/μl IGF-I treatment. Meanwhile, in theconcentration-response study, GH, insulin and high concentration of IGF-I and IGF-IIincreased IGFBP-3mRNA levels in the primary cultured hepatocytes. Morever, theenhancement effect of GH was in a time-dependent manner and reached maximallevels at48h treatment. The expression level of IGFBP-3mRNA in treated cells wassignificantly up-regulation and then bellowed the control level after10μM insulin and100ng/μl IGF-I treatment.（5）Using genome walking, he promoter sequences of Japanese flounder IGFBP-2a,-2b,-3and-4were obtained. The length was2145bp,1638bp,1811bp and2753bprespectively. Using several online prediction software, the cis-elements and potential transcription factor (TF) binding sites were determined. We only found the typicalTATA box (the sequence TATAAA) located30bp upstream of the putativetranscription start site (TSS) in Japanese flounder IGFBP-3and-4. Following theposition of TSS, several putative binding sites for TF and nuclear receptors wereidentified in the4promoters, including C/EBP, CREB, GATA, HNF, Pit-1, Oct-1,RXR, GR, PR and SRY. The putative binding sites for ER was only found in IGFBP-4,while Sox-5was only found in IGFBP-3. Furthermore, the AhR binding site wasexisted in the IGFBP-4and-2s promoters. The presence of all these TF binding sitesin the promoter region of Japanese flounder IGFBPs suggested that the TF were notconsistent among the4genes, the expression of Japanese flounder IGFBPs wereregulated with different regulation mechanisms and had different biological functions.