Effects And Underlying Mechanisms Of Transforming Growth Factor-beta 3 And Bone Morphogenetic Protein-2 Genes On The Proliferation And Differentiation Of Bovine Preadipocytes

Adipose tissue consists of a huge number of adipocytes,and the proliferation and differentiation of preadipocytes determine the amount of adipose tissue deposition in the body.Appropriate intramuscular fat deposition is a key factor affecting beef quality and is closely related to the production efficiency of beef cattle.Therefore,further exploration of the function and underlying mechanism of genes related to adipocyte proliferation and differentiation would be vital to the development of beef cattle industry.It has been reported that transforming growth factor-beta 3(TGF-β3)and bone morphogenetic protein-2(BMP-2)genes can participate in the regulation of cell proliferation and differentiation,but there are few studies on the effects of TGF-β3 and BMP-2 genes on bovine adipocyte proliferation and differentiation.Our previous study has shown that TGF-β3 and BMP-2 were highly expressed in bovine adipose tissue and mature adipocytes,and the expression levels of TGF-β3 and BMP-2 has changed significantly during the proliferation and differentiation of bovine preadipocytes.These results suggest that TGF-β3and BMP-2 may play an important role in the proliferation and differentiation of bovine preadipocytes.To further explore the cytological functions of TGF-β3 and BMP-2,primary preadipocytes derived from bovine subcutaneous adipose tissue were isolated and used as in vitro research materials.Its expression patterns were firstly examined during adipocytes maturation.Moreover,our research clarified the roles of TGF-β3 and BMP-2 in adipocyte proliferation and differentiation,and revealed the relevant molecular mechanisms of TGF-β3 and BMP-2 in regulating adipocyte proliferation and differentiation by using exogenous recombinant proteins,specific receptor inhibitors and small interfering RNAs(siRNAs),etc.Our findings would help to provide a theoretical basis for regulating fat deposition.This study was divided into five parts:1.Analysis of expression patterns of TGF-β3 and BMP-2 genes in different tissues and adipocytes of bovineIn order to explore the expression patterns of TGF-β3 and BMP-2 genes in different tissues and adipocyte differentiation,four 24-month-old Simmental bulls were selected as experimental animals in this chapter.Intramuscular fat,subcutaneous fat,heart,liver,spleen,lung,kidney and muscle tissues were collected immediately after slaughter,and subcutaneous preadipocytes were isolated to establish an in vitro cell model.Real-time quantitative PCR(RT-q PCR),immunohistochemistry and oil red O staining were used to detect the expression of TGF-β3 and BMP-2 genes in different tissues and adipocytes and verify the reliability of the in vitro proliferation and differentiation cell model.The results showed that TGF-β3 and BMP-2 were expressed in all bovine tissues,and high expressed in subcutaneous adipose tissue.Meanwhile,Immunohistochemical staining showed that TGF-β3 and BMP-2 proteins were predominantly localized in the subcutaneous adipose tissue.The isolated primary preadipocytes grew well,with clear cell boundaries and fibroblast-like morphology.At day 8 of induction,a large number of lipid droplets were observed after oil red O staining,indicating that preadipocytes had differentiated into mature adipocytes.During adipogenic differentiation,TGF-β3 and its receptor genes were high expressed before induction treatment and peaked at day 0.After induction treatment,their expression levels decreased significantly and then remained a low expression levels(P< 0.01).BMP-2 and its receptor genes were highly expressed in the early and late stages of adipogenic differentiation,but low in the middle stage.In conclusion,this study successfully established an in vitro preadipocytes proliferation and differentiation model,and revealed the expression patterns of TGF-β3 and BMP-2 in different tissues and adipocytes during adipogenic differentiation,providing a basis for subsequent researches.2.Effects of TGF-β3 and BMP-2 recombinant proteins on proliferation and differentiation of bovine preadipocytesAdipogenic differentiation involves a complex multi-gene regulatory network.This study aimed to explore the roles of TGF-β3 and BMP-2 genes in the proliferation and differentiation of bovine preadipocytes.The bovine preadipocytes were treated with exogenous TGF-β3 and BMP-2 proteins,respectively.The effects of exogenous proteins treatment on the proliferation and differentiation of adipocytes were investigated by CCK8,RT-q PCR,Western blot and oil red O staining.The results showed that TGF-β3 or BMP-2recombinant protein treatment significantly promoted the proliferation of bovine preadipocytes(P < 0.01),and the optimal treatment concentrations were 10 ng/m L and 100ng/m L,respectively.TGF-β3 recombinant protein treatment in the early stage of adipogenic differentiation reduced the numbers of lipid droplets in adipocytes,decreased the lipid accumulation of adipocytes(P < 0.01),and markedly inhibited the expression levels of peroxisome proliferator-ctivated receptor gamma(PPARγ),CCAAT/enhancer binding protein alpha(c/EBPα)and fatty acid binding protein 4(FABP4)at the 24 h and day 8 of induction(P < 0.01).Moreover,BMP-2 recombinant protein treatment in the middle stage of adipogenic differentiation increased the numbers of lipid droplets in adipocytes,promoted the lipid accumulation of adipocytes(P < 0.01),and significantly increased the expression levels of PPARγ(P < 0.01)and FABP4(P < 0.05)at the day 8 of induction.Above results indicated that TGF-β3 could promote the proliferation and inhibit the differentiation of bovine preadipocytes,while BMP-2 could promote the proliferation and differentiation of bovine preadipocytes.3.Effects of TGF-β3 and BMP-2 knockdown on proliferation and differentiation of bovine preadipocytesTo further verify the roles of TGF-β3 and BMP-2 genes in the proliferation and differentiation of bovine preadipocytes.The siRNAs(siRNA3 and siRNA6)specifically knocked down TGF-β3 and BMP-2 genes had been screened,respectively.The effects of TGF-β3 and BMP-2 knockdown on the proliferation and differentiation of bovine preadipocytes were studied by CCK8 assay,RT-q PCR,Western blot and oil red O staining.The results showed that the m RNA(P < 0.01)and protein expression(P < 0.01)levels of TGF-β3 and BMP-2 were significantly reduced in bovine preadipocytes which transfected with siRNA3 or siRNA6,establishing a reliable interference model.TGF-β3 or BMP-2knockdown increased the proportion of bovine preadipocytes in G1 phase(P < 0.01),inhibited the cell cycle transition from G1 phase to S phase,and significantly inhibited proliferation of bovine preadipocytes(P < 0.05).In addition,TGF-β3 knockdown markedly increased the numbers of lipid droplets and lipid accumulation in adipocytes(P < 0.01)as well as the expression levels of FABP4(P < 0.05)at the 24 h and c/EBPα(P < 0.01)at day8 of induction,promoting adipocyte differentiation.However,BMP-2 knockdown significantly reduced the numbers of lipid droplets and lipid accumulation in adipocytes(P< 0.01),and showed an inhibitory effect on adipocyte differentiation.Taken together,TGF-β3 or BMP-2 knockdown inhibited the proliferation of preadipocytes,while TGF-β3knockdown promoted and BMP-2 knockdown inhibited adipocyte differentiation,which was consistent with the results in Chapter 2.4.Effects of TGF-β3 and BMP-2 on proliferation and differentiation of bovine preadipocytes by regulating Smad signaling pathwayTGF-β3 and BMP-2 usually regulate downstream related responses by activating drosophila mothers against decapentaplegic protein(Smad)2/3 and Smad1/5/9 signaling pathways.This study aimed to investigate whether Smad2/3 and Smad1/5/9 signaling pathways are involved in the regulation of TGF-β3 and BMP-2 on the proliferation and differentiation of bovine preadipocytes,and to preliminarily explore the interaction between TGF-β3 and BMP-2.Bovine preadipocytes were treated with TGF-β3 and BMP-2recombinant proteins,respectively.Western blot results showed that the phosphorylation levels of Smad2 and Smad3 proteins were significantly increased after TGF-β3 treatment for 30 min and 1 h in bovine preadipocytes(P < 0.01),while treatment for 15 min only significantly increased the phosphorylation level of Smad3 protein(P < 0.01).After BMP-2treatment for 15 min,30 min and 1 h,the phosphorylation level of Smad1/5/9 protein was significantly increased(P < 0.01).Bovine preadipocytes were pretreated with TGF-β3 or BMP-2 receptor inhibitors for 2 h,and then treated with TGF-β3 or BMP-2 recombinant proteins for 1 h.Western blot results showed that 10 μM SB525334 could completely block the activation of Smad2 and Smad3 proteins in response to TGF-β3,and 0.5 μM or 5 μM LDN-193189 could completely block the activation of Smad1/5/9 in response to BMP-2.After blocking the TGF-β3/Smad and BMP-2/Smad signaling pathways with receptor inhibitors,the results of CCK8 and RT-q PCR assays showed that 10 μM SB525334 and 0.5μM LDN-193189 treatment completely blocked the pro-proliferative effects of TGF-β3 and BMP-2 on bovine preadipocytes.Compared with the blank control group,10 μM SB525334 treatment significantly increased the expression levels of FABP4 at the 24 h and day 8 of induction(P < 0.05)as well as blocked the inhibitory effect of TGF-β3 on the expression levels of PPARγ at day 8 of induction in bovine adipocytes.0.5 μM LDN-193189 treatment significantly decreased the expression levels of PPARγ,c/EBPα and FABP4 at day 8 of induction in bovine adipocytes(P < 0.05).Similarly,LDN-193189 treatment blocked the promoting effect of BMP-2 on the expression levels of PPARγ,c/EBPα and FABP4 at day 8 of induction.In addition,bovine preadipocytes were treated with TGF-β3 and BMP-2 recombinant proteins,respectively.The results showed that BMP-2 recombinant protein treatment significantly reduced the expression levels of TGF-β3 in adipocytes(P < 0.01),and TGF-β3 recombinant protein treatment significantly decreased the expression levels of BMP-2 and BAMBI in adipocytes(P < 0.01).In conclusion,TGF-β3 and BMP-2 regulate the proliferation and differentiation of bovine preadipocytes through Smad signaling pathway.TGF-β3 and BMP-2 exhibit mutual inhibitory effect.5.Effects of TGF-β3 on proliferation and differentiation of bovine preadipocytes by regulating MAPK signaling pathwayTGF-β family proteins can simultaneously activate the Smad signaling pathway and the Mitogen-activated protein kinase(MAPK)signaling pathway.Whether TGF-β3regulates the proliferation and differentiation of bovine preadipocytes via the MAPK signaling pathway is still unclear.In order to explore the role of MAPK signaling pathway in the process of adipocyte proliferation and differentiation in response to TGF-β3,CCK8 assay,RT-q PCR and Western blot were used to investigate the activation of MAPK signaling pathway by TGF-β3 treatment as well as the effect of down-regulated Smad2,Smad3 or MAPK signaling pathway on the proliferation and differentiation of bovine preadipocytes.The results showed that TGF-β3 treatment for 15 min,30 min and 1 h significantly increased the phosphorylation level of c-Jun N-terminal kinase(JNK)(P <0.01),and treatment for 30 min and 1 h markedly increased the phosphorylation level of p38 in bovine preadipocytes(P < 0.01).TGF-β3 treatment for 15 min,30 min,and 1 h did not significantly change the phosphorylation level of extracellular regulated protein kinase(ERK)1/2(P > 0.05),indicating that TGF-β3 can phosphorylate and activate JNK and p38 signaling pathways.However,SB525334 treatment blocked the activation of JNK and p38 signaling pathways in response to TGF-β3.To further clarify the effect of down-regulated Smad2,Smad3,JNK and p38 signaling pathways on the proliferation and differentiation of bovine preadipocytes.The experiment was divided into 6 groups: NC group,NC+TGF-β3group,si-Smad2+TGF-β3 group,si-Smad3+TGF-β3 group,si-JNK+TGF-β3 group and si-p38+TGF-β3 group.The results showed that the proliferation of NC+TGF-β3 group was significantly higher than that of NC group(P < 0.05).Compared with NC+TGF-β3 group,the proliferation of si-Smad2+TGF-β3 group,si-JNK+TGF-β3 group and si-p38+TGF-β3group was significantly decreased(P < 0.05),in which si-P38 +TGF-β3 group had the greatest reduction.About differentiation,the expression levels of PPARγ,c/EBPα and FABP4 in NC+TGF-β3 group were significantly higher than those in NC group(P < 0.05).Compared with the NC+TGF-β3 group,the expression levels of PPARγ in the si-Smad3+TGF-β3 group was significantly increased(P < 0.05),the expression levels of c/EBPα in the si-Smad2+TGF-β3 group was significantly decreased(P < 0.05),and the expression levels of c/EBPα in the si-p38+TGF-β3 group was significantly increased(P <0.05).In summary,TGF-β3 could activate JNK and p38 signaling pathways;Smad2,JNK and p38 signaling pathways were involved in the pro-proliferative effect of TGF-β3;Smad3and p38 signaling pathways mediated the inhibitory effect of TGF-β3 on differentiation of bovine preadipocytes.