Integrin α6β4 signaling has been shown to be ligand-independent in several model systems where the integrin does not require exogenous ligand to mediate its effects7. In our previous studies, we have found that exogenous laminin is not required to see changes in gene expression associated with enhanced invasion and migration13. Interestingly, the Jones group has demonstrated that integrin α6β4 promotes secretion and deposition of laminin-5, a major laminin isoform implicated in integrin α6β4 signaling, in the extracellular matrix, which in turn promotes motility of keratinocytes28. To test if endogenous secretion of laminin-5 is responsible for ligating integrin α6β4 in the Panc1 model, we plated Panc1-2G6 (low α6β4) and Panc1-3D7 (high α6β4) cells were plated onto collagen-coated coverslips for 4 hours and stained for the laminin γ2 subunit, which is unique to laminin-5. We find that in integrin α6β4 high expressing Panc1-3D7 cells, there is enhanced deposition of laminin, which strongly colocalizes with the integrin β4 subunit (Fig. 1A), which is in line with observations from keratinocyte studies. In contrast, cells with low integrin α6β4 have low detectable levels of laminin (Fig. 1D), which is not deposited extracellularly (Fig. 1B), implicating a deficient integrin α6β4 signaling network. Likewise, our previous studies demonstrate the enhanced migratory abilities of integrin α6β4 high versus low expressing pancreatic cancer cell lines when plated on laminin29. We chose to use these stable subpopulations derived from the Panc1 cell line as our model system for studying the impact of integrin α6β4 on the transcriptome as we have clearly demonstrated both variable levels of the integrin α6β4 and its cognate ligand, laminin-5.Figure 1Integrin α6β4 binds to and promotes secretion of laminin-5 in Panc1 cells. (A–C) Panc1 clones 3D7 (A; high α6β4) and 2G6 (B; low α6β4) were plated on collagen I coated coverslips and allowed to adhere under normal culture conditions for 4 hours. Cells were fixed and immunostained for integrin β4 subunit (red), laminin-5 (γ2 subunit; green), or f-actin (blue) as described in the Methods section. Cells were imaged by TIRF microscopy using the same exposure times and settings. Pearson’s coefficient for colocalization between integrin β4 and laminin-5 were 0.8 for Panc1-3D7 (A) and 0.011 for Panc1-2G6 (B). These values are representative for the 30 cells analyzed for each cell line. (C) Represents a rare β4 expressing cell in the Panc-2G6 cell population. (D) Western blot analysis of whole cell extracts from MDA-MB-231 (positive control), Panc1-2G6 and Panc1-3D7 cells for integrin β4, laminin-5 and actin (loading control).