CTSB Promotes Porcine Preadipocytes Differentiationby Degrading Fibronectinand Attenuatingthe Wnt/β-catenin Signaling Pathway

Obesity results from the overmuch fat deposition, which is likely to cause diseases suchas cancer, angiocardiopathy, and type II diabetes. Fat deposition results from the increase ofadipocyte numberand size, which was regulated by several factors. Adipose tissue is not onlya energy storage organ but also an important secretory organ in the body. Cathepsin is asecretory protein that secretes by lysosome. There are about15members in its family andmany of them play an important role during adipocytes differentiation. However, as a memberof cathepsin family, the role of CTSB during adipogenesis is unclear. Thus, we used theporcine preadipocytes as experimental material of this study, and then explore theregulatorymechanism of CTSB during preadipocytes differentiation. Futhermore, as CTSBcould degrade fibronectiin and there are several reports showing that fibronectin, which is animportant component of extracellular matrix, affects adipocytes differentiation by changingthe structure and surface receptors of adipocytes. As it is a target gene of Wnt/β-cateninsignaling, we speculated CTSB regulates adipogenesis by Wnt/β-catenin signaling. Hence, byusing knockdown or ovexpression to decrease or increase the expression of CTSB then weused the Oil red staining, real-time quantification PCR and western blot to illuminate themechanism of CTSB during porcine preadipocyetes differentiation.The results as follow:1. CTSB gene was expressed in various tissues of3day-old piglets and180day-old pigs, itwas abundant in kidney and liver and moderate in adipose tissue. The expression of CTSBwas higher in pocine preadipocytes than mature adipocytes. The level of CTSB increasedduring adipogenesis.2.The adenovirus vector of CTSB overexpression was constructed, then high titer virus wasaquired after packaging and propagating. The differentiation was promoted in the adipocyetesthat infected by Ad-CTSB. Moreover, the lipid accumulation and the expression of adipogenicgenes increased.3. Constructed the lentiviral vector of CTSB interference, then aquired high titer virus after packaging and chose the most high interference-efficient shRNA, of which the interferenceefficiency was above80%. The differentiation was repressed in the adipocyetes that infectedby sh-CTSB. Moreover, the lipid accumulation and the expression of adipogenic genesdecreased.4. The detection of immunofluorescence showed that fibronectin in the cell surface decreasedsignificantly in cells infected with Ad-CTSB. Collected the culture medium during adipocytesdifferentiation then detected by western blot, we found that degradation debris of fibronectinincreased in Ad-CTSB group compared with control. This result suggested that CTSBpromoted porcine differentiationg by degrading fibronectin.5. CTSB overexpression reduced the expression of β-catenin and CTSB knockdown increasedthe expression of β-catenin. Overexpressed CTSB in cells that treated with LiCl relieved theanti-adipogenic effect of LiCl and reduced the nuclear translocation of β-catenin.The above resultsdemonstrated that CTSB facilitated preadipocytes differentiation, whichfunctioned by degrading fibronectin and inhibited the nuclear translocation of β-catenin.These data provides a theoretical basis to improve the molecular mechanism of cathepsinfamily during adipocytes differentiation and treatment of obesity and its related diseases.