Abstract
Purpose: :
Syndecan 1 (Sdc1) is a heparan sulfate proteoglycan that mediates the activation of integrins and modulates TGFβ1 signaling and cell migration. To study the role of Sdc1 in mediating TGFβ1 signaling, we set out to generate a human corneal epithelial cell line in which expression of Sdc1 is suppressed.
Methods: :
Several different silencing sequences were tested for their ability to reduce expression of Sdc1 in the human corneal limbal epithelial (HCLE) cell line using transient transfection. The sequence which functioned to reduce Sdc1 expression most was used to generate ShRNA packaged into lentiviral particles, as a control, we used lamin ShRNA. Immunoblots were used to confirm the reduction in expression of Sdc1. Parental HCLE cells, HCLE cells transfected with the Shlamin lentivirus and HCLE cells transfected with ShSdc1 lentivirus were used to determine the impact of the reduction of sdc1 expression on cell migration, TGF β1 signaling and ECM assembly. The rate of migration for the three cell lines was assessed by time-lapse microscopy using Metamorph software for image analysis. TGFβ1 signaling was assessed by treating cells with 0.25ng/ml of TGFβ1 and assessing Smad2 phosphorylation over time. ECM matrix preparations were prepared for all three lines and laminin332 and fibronectin assembly into ECM were evaluated.
Results: :
Sdc1 expression was reduced in the ShSdc1 cells compared to the ShLamin and parental HCLE cells. The rate of cell migration of ShSdc1 HCLE cells was significantly reduced compared to parental and control cells. TGFβ1 treatment induced an increase in pSMAD2 expression within 15min in all three cell lines. However the increase in pSMAD2 was greater in the ShSdc1 cells. Cells expressing reduced levels of Sdc1 were found to deposit less fibronectin and less LN332 compared to ShLamin cells. Interestingly, both lentiviral infected cell lines showed altered ECM accumulation compared to the parental HCLE cells.
Conclusions: :
As seen in primary mouse epidermal keratinocytes lacking Sdc1, human corneal epithelial cells expressing less Sdc1 migrate slower and show altered TGFβ1 induced signaling. The ShSdc1 HCLE cells also show evidence of altered matrix assembly compared to control cells transfected with ShLamin lentivirus. However, our results raise concern over the impact of lentiviral infection on matrix assembly since the parental cells show differences in matrix assembly compared to cells infected with either lentivirus.
Keywords: cornea: epithelium • wound healing • extracellular matrix