The Effect Of Biglycan On Cytokine Signaling In Bovine IVD Cells

Objective:Low back pain(LBP) is ubiquitous,and its social economic effects are enormous.Inter vertebral disc(IVD) degeneration is thought to be the main reason of LBP and IVD herniation.The degeneration of an IVD may result from an imbalance between the anabolic and catabolic processes or the loss of steady state metabolism that is maintained in the normal disc.Alterations in both anabolic and catabolic processes are thought to play key roles in the onset and progression of IVD egeneration, but the biochemical processes that regulate these changes are poorly understood.The anabolic regulators include polypeptide growth factors, such as EGF,transforming growth factor(TGF),and the bone morphogenetic proteins(BMPs,including OP-1).Interleukin-1(IL-1),a proinflammatory cytokine,is thought to contribute significantly to the loss of matrix homeostasis during IVD disease.Those cytokines may have a fundamental role in the maintenance of normal disc homeostasis and be involved in regenerative or reparative responses in the early stage of degenerative disc disease.Biglycan is a member of the family of small leucine rich proteoglycans(SLRP).Biglycan interacts with several collagens and regulates the activity of some cytokines.Its expression and biological activity could also be modulated by some cytokines in the extracellular matrix(ECM).The objectives of this study are as below:1. To evaluate if Biglycan can affect cytokines(EGF,OP-1,and IL-1) on their signaling in bovine IVD cells;2.If it does,then,is there any deference between anulus fibrosus(AF) and nucleus pulposus(NP) cells.Methods:IVDs were isolated from the tails of young adult(15- to 18-mo-old) bovine purchased at a local slaughterhouse.The initial four to five levels of IVDs(coccygeal 1-4) from bovine tails were isolated,separated into the NP and the outer two-thirds of the AF,and separately pooled for each experiment.And then the cells were cultured in mono-layer as previously described for 3 to 7 days.1.First,we planed to observe the effect of Biglycan alone on bovine IVD cells(with a focus on the MAPK/ERK signaling pathway). The cells were serum starved overnight prior to treatment.Then,the cells were treated with Biglycan as these 3 aspects.1).In order to find the optimal time point for Biglycan's effect,a Biglycan time-curve was performed.2).Similar to the time curve,a Biglycan concentration curve was done to find the optimal concentration for Biglycan's effect.3).A special inhibitor to the Erk pathway was used to determine specificity of Biglycan signal in Erk pathway.Following the above 3 different treatment,the cells were lysed with cell lysis buffer containing PMSF and phosphate inhibitor cocktail to prevent phosphatase enzyme activity. Protein concentration was determined by the BCA assay to ensure equal gel loading.Western blots were then performed,using the pERK antibodies."tERK" antibody was used to normalize the signal of pERK.Read the film density to get amount of aimed protein,and Analyses the data.2.The 2nd major step is to observe the effect of growth factors/cytokines when used alone to IVD cells(MAPK/ERK signaling pathway).Same as the first experiment,the cells were serum starved overnight prior to treatment.Then,the cells were treated with EGF, OP-1,and IL-1 respectively.Following these treatment,the cells were lysed with cell lysis buffer containing PMSF and phosphate inhibitor cocktail to prevent phosphatase enzyme activity.Protein concentration was determined by the BCA assay to ensure equal gel loading.Western blots were then performed,using the pERK antibodies."tERK" antibody was used to normalize the signal of pERK.Read the film density to get amount of aimed protein,and Analyses the data.3.The 3^rd major step is the core experiment of this study.The aim of this step is to identify if Biglycan has an effect on growth factor/cytokine signaling.The BAF and BNP cells were pretreated with Biglycan at various concentrations for five minutes.After this pretreatment,the growth factor or cytokine was added to the cells for 10 minutes,which was the optimal time point for Biglycan effect based upon the 1^st step---Biglycan time curve experiment.Of course,during this step,the various concentration of Biglycan included the optimal concentration which was also from 1^st step Biglycan concentration curve experiment.After the ten minute combination treatment of Biglycan and cytokines or growth factors,the media was removed,the cells were washed with PBS,and they were lysed.Then underwent BCA assay, Western Blot,and so on.This step also included 3 different experiments: one was combination treatment of Biglycan and EGF,to identify if Biglycan has an effect on EGF signaling;One was combination treatment of Biglycan and OP-1,to identify if Biglycan has an effect on OP-1 signaling;The other was combination treatment of Biglycan and IL-1,to identify if Biglycan has an effect on IL-1 signaling.Each experiment was repeated 2-5 times in same condition,SD and p values were calculatedResults:1.Biglycan time-curve experimentFor the Biglycan time-curve experiment,we get the film density from western blots using the pERK and tERK antibodies,tERK was used to normalize the signal of pERK.Then compared the signal of Biglycan to the control.We found that Biglycan had the optimal effect at 10 min both in AF cells and NP cells while focus on ERk pathway.At this time point,Biglycan upregulate the signal 3-4 fold to the control. The difference is statistically significant.The difference between AF and NP wasn't significant.2.Biglycan concentration-curve experimentSimilar to the Biglycan time-curve experiment,Biglycan had the optimal effect at 20uM both in AF cells and NP cells while focus on ERk pathway.At this concentration point,Biglycan upregulate the signal 3-4 fold to the control.The difference is statistically significant.The difference between AF and NP wasn't significant.3.Inhibitor's effectSignaling got down significantly both in AF and NP cells treated with Erk inhibitor alone,a special inhibitor for ERk pathway.Similarly, both AF and NP cells treated with the combination of Erk inhibitor and Biglycan,the signaling also got down significantly,while Biglycan upregulate the Erk pathway signaling significantly.But,when look at PI3K inhibitor,it had no effect on Biglycan signaling in Erk pathway, because it is not an ERk pathway special inhibitor.The difference between AF and NP wasn't significant.4.Cytokines effect on IVD cells signalingIn these experiments,our data showed that EGF,OP-1,and IL-1 all can up-regulate IVD ceils signaling in Erk pathway.EGF had strong signal in Erk pathway,specific pathway for EGF.Although main pathways for OP-1 and IL-1 are pSMAD and pNFKB separately,they also showed partial effect on ERk pathwy.The difference between AF and NP wasn't significant.5.Biglycan's effect on cytokine signalingWhen combined different Biglycan concentration with EGF,OP-1, and IL-1 respectively to treat the IVD cells,cytokine treated alone had the highest effect on Erk pathway.As the concentration of Biglycan went up, the cytokine signal went down both in BAF and BNP cells.And the difference was significant.But he difference between AF and NP wasn't significant.Conclusion:1.Biglycan has the ability to bind to the IVD cells to activate the ERK pathway.This effect is time and concentration dependent.The optimal time and concentration for both BAF and BNP cells are 10 minute and 20 uM.Erk inhibitor can inhibit the Biglycan-generated signal specifically in ERk pathway.BAF and BNP had no significant difference.2.In agreement with published literature,our results also showed EGF,and OP-1 promote matrix synthesis,therefore those cytokines contribute to the cell's repair ability.IL-1 is believed to play a critical role in the induction of enzymes that destroy proteoglycans and other negative function.When combined with cytokines,as our results showed, Biglycan interferes with both EGF,OP-1,and IL-1 ERK signaling. Through this,Biglycan may regulate some of the effects of growth factors. When Biglycan is present in the degenerated IVD in high levels,this regulatory role of Biglycan may be very important to maintain the metabolism balance.In turn of this balance,Biglycan should be beneficial for the repair of IVD.There were no significant difference between BAF and BNP.