The Role And Mechanisms Of Spectrin-βⅡ In The Differentiation Of Adipocytes And Osteoblasts

BackgroundsObesity and osteoporosis are two of the most common health problems in the elderly population.According to incomplete statistics,more than 600 million people worldwide suffer from obesity and more than 200 million people suffer from osteoporosis.Adipocytes and osteoblasts originate from a common precursor,the multipotent mesenchymal stem cells(MSCs),which also give rise to other lineages including chondrocytes and myocytes.Importantly,between adipogenic and osteogenic lineage commitment and differentiation,differentiation of MSCs into one lineage will inhibit their differentiation toward the other lineage.This balance is regulated by numerous signaling pathways.The balance is tilted toward adipogenesis with age,obesity,and in several bone diseases with progressive bone loss such as osteoporosis.Therefore,exploring the molecular mechanisms that affect adipocyte and osteoblast differentiation will provide new therapeutic strategies for the prevention and treatment of obesity and osteoporosis.Spectrin-βⅡ is a non-erythrocyte membrane spectrin,which is widely present in various tissues and cells,and plays an important role in maintaining cell morphology,cell polarity,cell membrane stability,and cell-to-cell interactions.The precursor mRNA of spectrin-βⅡ is regulated by alternative splicing,and mainly produces two different mRNA variants,which respectively encode two protein isoforms:Spectrin-βⅡ Σ1 and Spectrin-βⅡ Σ2.Studies have shown that the RNA-binding protein QKI plays an important role in promoting adipocyte differentiation and in inhibiting osteoblast differentiation.Our previous studies found that QKI regulates the alternative splicing of Spectrin-βⅡ and promotes the expression of the Spectrin-βⅡ Σ2.Studies have shown that Spectrin-βⅡ inhibits liver cancer by regulating the canonical Wnt/β-catenin pathway,which is regarded as the master moderators of adipogenesis and osteoblastogenesis.Moreover,QKI inhibits colon cancer through the canonical Wnt/β-catenin pathway.Then,can Spectrin-βⅡ and its two main isoforms,Spectrin-βⅡ Σ1 and Spectrin-βⅡ Σ2,participate in adipocyte and osteoblast differentiation by regulating the canonical Wnt/β-catenin pathway? There are no reports in the literature up to now.Objectives1.To analyze the expression patterns of Spectrin-βⅡ,Spectrin-βⅡ Σ1 isform and Spectrin-βⅡ Σ2 isform during the adipocyte differentiation and osteoblast differentiation.2.To investigate the effects of Spectrin-βⅡ,Spectrin-βⅡ Σ1 isform and Spectrin-βⅡ Σ2 isform on adipocyte differentiation and osteoblast differentiation.3.To reveal the mechanis how Spectrin-βⅡ,Spectrin-βⅡ Σ1 isform and Spectrin-βⅡ Σ2 isform regulate adipocyte differentiation and osteoblast differentiation.Methods1.For adipocyte differentiation,multipotent C3H10T1/2 cells were cultured in the classical adipocyte differentiation medium for 8 days and stained with Oil Red O staining.For osteoblast differentiation,multipotent C3H10T1/2 cells were cultured in the classical osteoblast differentiation medium for 21 days and stained with Alizarin red staining.Western blot and Q-PCR were used to detect the expressions of Spectrin-βⅡ,Spectrin-βⅡ Σ1,Spectrin-βⅡ Σ2 and adipogenic genes during adipocyte differentiation,and to detect the the expression of Spectrin-βⅡ,Spectrin-βⅡ Σ1,Spectrin-βⅡ Σ2 and osteogenic genes during osteoblast differentiation.2.The C3H10T1/2 cells were infected with lentiviruses expressing Spectrin-βⅡ-specific,Spectrin-βⅡ Σ1 isform-specific,Spectrin-βⅡ Σ2 isform-specific hairpin sh RNA sequence or control sh-scramble sequence to stably knock down Spectrin-βⅡ or each isoform individually.Then,the stable cell lines were differentiated into adipocyte and osteoblasts respectively.Adipocytes were detected by Oil Red O staining and osteoblasts were detected by Alizarin red staining.The expression of adipogenic genes were determined by Western blot and Q-PCR.The expression of osteogenic genes were determined by Q-PCR.3.C3H10T1/2 cells were infected with the Synergistic activation mediator to stably endogenously overexpress Spectrin-βⅡ Σ1 or Spectrin-βⅡ Σ2,and then differentiated into adipocyte and osteoblasts respectively.Adipocytes were detected by Oil Red O staining and osteoblasts were detected by Alizarin red staining.The expression of adipogenic genes were determined by Western blot and Q-PCR.The expression of osteogenic genes were determined by Q-PCR.4.To investigate mechanisms whereby Spectrin-βⅡ,Spectrin-βⅡ Σ1 and Spectrin-βⅡ Σ2 regulate cell fate,total RNA was extracted from Spectrin-βⅡ-specific knockdown,Spectrin-βⅡ Σ1 isform-specific knockdown,Spectrin-βⅡ Σ2 isform-specific knockdown or control C3H10T1/2 cells,and then,all samples were analyzed using RNA-Seq.5.According to RNA-Seq analysis,Spectrin-βⅡ Σ1 isform-specific knockdown increased sfrp2 expression,while Spectrin-βⅡ Σ2 isform-specific knockdown reduced sfrp2 expression in C3H10T1/2 cells,indicating sfrp2 is responsible for the effects of Spectrin-βⅡ Σ1 and Spectrin-βⅡ Σ2 on adipocyte and osteoblast differentiation.To verify this hypothesis,we knocked down sfrp2 in Spectrin-βⅡ Σ1 isform-specific knockdown C3H10T1/2 cells,whereas overexpressed sfrp2 in Spectrin-βⅡ Σ2 isform-specific knockdown C3H10T1/2 cells by infecting lentivirus to detect the changes in adipocyte and osteoblast differentiation.6.We detected β-cantenin phosphorylation in Spectrin-βⅡ-specific knockdown,Spectrin-βⅡ Σ1 isform-specific knockdown,Spectrin-βⅡ Σ2 isform-specific knockdown,Spectrin-βⅡ Σ1 isform-specific knockdown meanwhile sfrp2 knockdown,and Spectrin-βⅡ Σ2 isform-specific knockdown meanwhile sfrp2 overexpressed C3H10T1/2 cells by Western blot.Results1.The mRNA and protein levels of Spectrin-βⅡ,Spectrin-βⅡ Σ1,and Spectrin-βⅡ Σ2were gradually increased during adipocyte and osteoblast differentiation.Moreover,the expression of adipogenic genes(PPARγ,C/EBPα,FABP4)elevated during adipocyte differentiation,while the expression of osteogenic genes(Runx2,Osterix,and OCN)increased during osteoblast differentiation.2.The sh RNA-mediated knockdown of Spectrin-βⅡ had little effect on adipocyte and osteoblast differentiation.Interestingly,the isoform-specific sh RNA that target Spectrin-βⅡ Σ1 inhibit adipocyte differentiation and promoted osteoblast differentiation,while the isoform-specific sh RNA that target Spectrin-βⅡ Σ2 promoted adipocyte differentiation and inhibit osteoblast differentiation.Moreover,the isoform-specific sh RNA that target Spectrin-βⅡ Σ1 increased the expression of Spectrin-βⅡ Σ2.3.Over-expression of Spectrin-βⅡ Σ2 promoted adipocyte differentiation and inhibit osteoblast differentiation.4.RNA-Seq analysis showed that Spectrin-βⅡ Σ1 isform-specific knockdown increased sfrp2 expression,while Spectrin-βⅡ Σ2 isform-specific knockdown reduced sfrp2 expression in C3H10T1/2 cells.Western blot and Q-PCR tests confirmed the above results.5.The phosphorylation of β-cantenin was significantly increased in Spectrin-βⅡ Σ1isform-specific knockdown C3H10T1/2 cells,whereas the phosphorylation of β-cantenin was significantly decreased in Spectrin-βⅡ Σ1 isform-specific knockdown C3H10T1/2cells compared to control cells.6.Knockdown sfrp2 in Spectrin-βⅡ Σ1 isform-specific knockdown C3H10T1/2cells reduced the elevated phosphorylation level of β-catenin,and partially restored the promoted adipogenesis and the inhibited osteogenesis.Overexpressing sfrp2 in Spectrin-βⅡ Σ2 isform-specific knockdown C3H10T1/2 cells increased the declined phosphorylation level of β-catenin and recovered the inhibited adipogenesis and the promoted the promoted.Conclusions1.Spectrin-βⅡ Σ1 inhibits adipocyte differentiation while promotes osteoblast differentiation;Spectrin-βⅡ Σ2 promotes adipocyte differentiation while inhibits osteoblast differentiation.The effect of two isforms are opposite.2.The mechanism is that Spectrin-βⅡ Σ2 inhibits the Wnt/β-catenin signal pathway by upregulating sfrp2 expression;Spectrin-βⅡ Σ1 promotes the Wnt/β-catenin signal pathway by downregulating sfrp2 expression.3.Spectrin-βⅡ Σ1 can inhibit the expression of Spectrin-βⅡ Σ2,and its down-regulation on sfrp2 may be achieved by reducing the expression of Spectrin-βⅡ Σ2.