gms | German Medical Science

Objectives: In mammals, hyaluronan (HA) is formed by three known hyaluronan synthases (HAS1, HAS2 and HAS3), which are complex transmembrane proteins. HA seems to regulate the bone remodelling process by affecting the properties of mesenchymal stem cells (MSCs), osteoblasts and osteoclasts. While HAS activity depends on a localization in the plasma membrane, the mechanisms regulating the intracellular transport towards the plasma membrane are hardly understood. Due to the fact that an interaction with the cytoskeleton is regulating other members of the same enzyme family, specifically chitin synthases and cellulose synthases, we hypothesize an interaction of HAS and actin filaments and furthermore the mechanotransduction of the synthases' expression, transport and/or activity.

Methods: We generated immortalized human mesenchymal stem cells (hMSCs, SCP-1) constitutively expressing eGFP-tagged HAS by lentiviral gene transfer (SCP1-HAS1-eGFP, SCP1-HAS2-eGFP and SCP1-HAS3-eGFP). The expression of the transgene HAS was analyzed by RT-PCR, western blot, FACS analysis and direct fluorescence microscopy or immunofluorescence. hMSCs expressing lifeact-RFPruby and HAS-eGFP were investigated in a video timelapse analysis in order to study the putative interaction of HAS-eGFP with the actin cytoskeleton. In addition we investigated the impact of shear stress on hMSCs under defined flow conditions in cell culture. Therefore the change at the mRNA level mRNA of the expression of all three HAS isoforms was analyzed by quantitative real time RT-PCR after exposure to the stimulus.

Results and Conclusion: hMSCs cultured under shear stress showed an upregulation of HAS expression on the mRNA level. Transgene HAS expression was verified on mRNA and protein level. The enzymatic activity of the transgene HAS was determined by HA-ELISA and by staining of HA. The HAS-eGFP proteins are globular structured and aligned along the actin filaments. The timelapse pictures show that the HAS-eGFP moves without loss of their alignment to actin.

Here, we were able to show the regulation of HAS expression via mechanotransduction. In addition, we generated hMSCs expressing eGFP tagged HAS in their active form. Furthermore, we have first hints for an interaction of the transgene HAS with the actin cytoskeleton. At the moment, we analyze, if HAS activity and their transport towards the plasma membrane are changed by shear stress. These interaction and possible changes in the synthases' location due to mechanical stimuli could be analyzed by exposing the transgenic cells to shear stress. Our cells can be used for further investigation of the functional and regulatory role of HAS in the bone microenvironment.