Efficient TGFβ-induced epithelial-mesenchymal transition depends on hyaluronan synthase HAS2

Epithelial-mesenchymal transition (EMT) is a developmental program, which can be adopted by cancer cells to increase their migration and ability to form metastases. Transforming growth factor beta (TGF beta) is a well-studied inducer of EMT. We demonstrate that TGF beta potently stimulates hyaluronan synthesis via upregulation of hyaluronan synthase 2 (HAS2) in NMuMG mammary epithelial cells. This stimulatory effect requires the kinase active type I TGF beta receptor and is dependent on Smad signaling and activation of the p38 mitogen-activated protein kinase. Knockdown of HAS2 inhibited the TGF beta-induced EMT by about 50%, as determined by the phase contrast microscopy and immunostaining using the EMT marker ZO-1. Furthermore, real-time PCR analysis of the EMT markers fibronectin, Snail1 and Zeb1 revealed decreased expressions upon HAS2 suppression, using specific small interfering RNA (siRNA) for HAS2. Removal of the extracellular hyaluronan by Streptomyces hyaluronidase or inhibiting the binding to its cell surface receptor CD44 by blocking antibodies, did not inhibit TGF beta-induced EMT. Interestingly, HAS2 suppression completely abolished the TGF beta-induced cell migration, whereas CD44 knockdown did not. These observations suggest that TGF beta-dependent HAS2 expression, but not extracellular hyaluronan, has an important regulatory role in TGF beta-induced EMT.