Effect Of Cigarette Smoke Extract Or TGF-Beta1 On Hyaluronan Production And Hyaluronan Modulating Enzymes In Primary Murine Lung Fibroblasts

Hyaluronan (HA) is a component of the extracellular matrix and low molecular weight (LMW) HA fragments have pro-inflammatory capacities. Exposing mice to cigarette smoke (CS) for 1 or 6 months results in enhanced deposition of LMW HA in lung parenchyma and airway walls and in altered expression of HA synthases and hyaluronidases (Bracke et al ., Am J Respir Cell Mol Biol. 2010;42(6):753-61). To pinpoint a source of HA, we studied HA-production and expression of HA modulating enzymes in primary murine pulmonary fibroblasts stimulated with cigarette smoke extract (CSE) or TGF-β1. Fibroblasts were isolated from lungs of C57BL/6 mice and cultured in vitro . At passage 6, cells were stimulated for 24h or 48h with control medium, 5% CSE or 2ng/ml TGF-β1. mRNA expression of HA synthases (Has1, Has2, Has3) and hyaluronidases (Hyal1, Hyal2) was evaluated by RT-PCR. HA production was measured in supernatant by ELISA. In vitro stimulation of pulmonary fibroblasts with CSE significantly decreased the mRNA expression of Has1 (synthesizing high molecular weight (HMW) HA) and significantly increased the expression of Hyal2 (degrading HMW HA to LMW HA fragments). Stimulation with TGF-β1 resulted in significantly increased mRNA expression of Has2 (synthesizing HMW HA). Accordingly, HA-levels in the fibroblast supernatant decreased significantly upon 48h stimulation with CSE, while they were significantly increased upon 24h or 48h stimulation with TGF-β1. Decreased Has1 and increased Hyal2 in CSE-stimulated fibroblasts suggests reduced synthesis and enhanced breakdown of HMW HA. This may contribute to the accumulation of LMW HA fragments, observed in CS-exposed mice.