Investigating Compensation of Av Integrins in LX-2 Cells in Vitro

Abstract

Fibrosis is a leading cause of mortality worldwide, accounting for 45% of deaths in the industrial world. Fibrogenesis for multiple diseases and organs has been associated with the dysregulation of integrins and plays a role in different disease states. Integrins are cell adhesion molecules that reside on the surface of cells as transmembrane proteins made of an alpha and beta subunit. There are 24 known integrin molecules classified into different families depending on the subunits (i.e. αv family of integrins) or by the ligands they bind to (i.e. RDG binding family of integrins). They play an important role in many different vital cell processes including migration, proliferation, differentiation, and apoptosis. The integrins’ involvement in all of these processes can facilitate the communication between extracellular matrix, inflammatory cells, fibroblasts, and parenchymal cells through their ability to bind to extracellular matrix ligands, cell-surface ligands, and soluble ligands. Despite their critical role in fibrosis, integrin compensatory regulation is not fully understood. The objectives of this study were to characterize how the αv integrins regulate each other in both a normal and pro-fibrotic environment in order or decipher any mutual regulation between the αv integrins and to assess if there is a good in vitro model to look at potential compensation mechanisms specifically in an inflammatory environment. Antibody inhibitors and siRNA were used to manipulate the expression profiles of LX-2 cells that were incubated under normal culture conditions or after treatment with TGF-Β, a known ligand for the αv integrins and a pro-fibrotic cytokine, to mimic a pro-fibrotic environment in vitro. LX-2 cells are spontaneously immortalized human hepatic stellate cells and stellate cells are believed to play a role in fibrotic diseases of the liver. Cells were analyzed by qPCR and flow cytometry to assess the changes in the integrin expression profile. The integrin profile of LX-2 cells was not changed by antibody treatment targeting αvβ3 or αvβ5. Upon knockdown of β3 or β5 expression using small interfering RNA (si-RNA) treatment, a marked decrease in the cell surface expression of the αv subunit was observed without a change in the mRNA levels of the αv subunit. Knockdown of β3 expression using si-RNA treatment did not affect the cell surface expression of αvβ5 and knockdown of β5 expression using si-RNA treatment did not affect the cell surface expression of αvβ3. Our data suggests the αv subunit pairs mostly with β3 and β5 and that under these normal conditions the αv subunit is not re-pairing with other subunits when knocked down.