Twist1 Evokes Matrix Metalloproteinase 9 and Collagen IV Synthesis in Activated Pancreatic Stellate Cells

Abstract

Background: Pancreatic cells are embedded in an extracellular matrix (ECM) and are also surrounded by a thin sheet of specialized extracellular matrix known as basal lamina. In healthy pancreas, pancreatic stellate cells (PaSCs) are responsible for the synthesis of basal lamina, which is mainly composed of collagen IV and laminin. In chronic pancreatitis (CP), PaSCs are responsible for the synthesis of fibrotic tissue, which is mainly composed of fibronectin and collagen I. Reactive oxygen species (ROS), which are predominant inflammatory mediators in CP, evoke the formation of fibrotic tissue by PaSCs. One of the sources of ROS is NADPH oxidase (Nox) enzymes. In particular, Nox1 has been associated with both fibrogenesis in a CP mouse model and an up-regulation of the transcription factor Twist1 and matrix metalloproteinase (MMP) 9 in CP-activated PaSCs. I sought to determine the functional relationship between Twist1 and MMP-9, as well as other PaSC-produced proteins. Therefore, my aim was to assess the extent to which Twist1 up-regulates MMP-9 and other PaSC-produced proteins. Methods: To overexpress Twist1, I infected activated PaSCs from Nox1-null mice with retroviruses expressing either mouse Twist1 or the empty backbone. I compared the relative expression of MMP-9 and other PaSC-produced proteins using quantitative PCR. To examine whether MMP-9 expression is regulated by Twist1 at the transcriptional level, I carried out a dual-luciferase reporter assay using a pGL2 Luciferase Reporter Vector carrying the human MMP-9 (pGL2-hMMP-9) promoter. I co-transfected HEK293 cells with human Twist1 in pCMV6 vector, or the empty backbone (negative control) along with human pGL2-hMMP-9 promoter vector or pGL3-control vector using Lipofectamine 3000. As a positive control, I co-transfected HEK293 cells with human NF-ĸB1 in pFUW-tetO vector along with the pGL2-hMMP-9 promoter. After 48 h, I performed the dual-luciferase reporter gene assay. Results: I found that the up-regulation of Twist1 in culture-activated PaSCs from Nox1-null mice increased MMP-9 mRNA level, but it did not modify the expression of PaSC-produced proteins linked to fibrosis [e.g., collagen I, fibronectin, α-smooth muscle actin, transforming growth factor-β (TGF- β), and interleukin-6 (IL-6)]. Therefore, I studied the expression of collagen IV, a component of basal lamina and found that the expression of Twist1 in activated PaSCs from Nox1-null mice increased collagen IV at the mRNA level. I also found that Twist1 increased the expression of MMP-9 at the transcriptional level in a NF-ĸB dependent manner using a dual-luciferase assay. Conclusion: Twist1 in PaSCs induced the expression of MMP-9 and collagen IV, at the transcriptional level. NF-ĸB was required for the transcriptional up-regulation of MMP-9 by Twist1. The expression of other PaSC-produced proteins, including collagen I, fibronectin, IL-6, α-smooth muscle actin, and TGF-β, were not affected. Since Twist1 in activated PaSCs was responsible for the synthesis and remodeling of the basal lamina in healthy pancreas, I concluded that the overexpression of Twist1 using a retrovirus approach was not sufficient to change the phenotype of PaSC from a basal lamina “maker” to a fibrotic tissue “maker.”