Regulation of RHBDL2-Mediated Thrombomodulin Shedding and Its Role on Keratinocyte Migration

• Main Authors: Hung-yu Lin, 林宏栯
• Other Authors: Hua-lin Wu
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/43887761957885071389

碩士 === 國立成功大學 === 生物化學研究所 === 96 === Thrombomodulin (TM) is a type I transmembrane protein constitutively expressed in endothelial cells, and acts as a cofactor in natural protein C anti-coagulant system. TM is involved in several biological processes, including anti-coagulation cascade, inflammatory response, cell-cell adhesion as well as angiogenesis through interaction with diverse proteins. TM expression is also observed in the spinous layer of epidermis and highly expressed in the neoepidermis of wounded skin tissue. In previous studies, we demonstrated that the treatment of recombinant TM extracellular fragment elevated keratinocyte migration, indicating that TM might participate in epithelial dynamics. Moreover, a recent report revealed that TM transmembrane domain was cleaved by mammalian rhomboid-like serine protease (RHBDL2) which did not shed other EGF-like ligands as Drosophila rhomboid did. These findings implied that TM could be a physiological substrate for RHBDL2. However, the significance of RHBDL2-mediated TM ectodomain shedding in keratinocytes remains elusive. In this study, we observed that soluble TM fragments (sTM) collected from the culture medium of TM-abundant human epithelium cell line HaCaT cells had almost the same size as recombinant human TM extracellular fragment (TMD123). The release of sTM increased upon the stimulation of tumor necrosis factor alpha (TNF alpha) and epidermal growth factor (EGF), and specifically inhibited by a serine protease inhibitor 3, 4-dichloroisocoumarin (DCI). Moreover, extensive wounding could initiate proteolytic processing of TM in either Western blot or ELISA assay. The TM cleavage was not corresponded with the RNA expression of RHBDL2, but was susceptible to DCI treatment. Besides, we established RHBDL2- or active-site mutated RHBDL2 (RHBDL2-SA) stably expressed HaCaT cells. Substantially increased TM ectodomain shedding of RHBDL2-HaCaT cells was observed as determined by Western blot assay. The localization of RHBDL2 in HaCaT cells was found in cell membrane. RHBDL2-HaCaT cells displayed higher wound-closure percentage in comparison with corresponding controls (69.6 and 39.5% 24 hr after wounding), and this higher migratory ability was independent of cell proliferation during first 24 hours. However, higher proliferation rate was also observed in either RHBDL2- or RHBDL2-SA-HaCaT cells after 48 hr. In cell-matrix adhesion assay, RHBDL2- HaCaT cells also possessed less ability to adhere to type I collagen. In conclusion, we propose that TM shedding mediated by RHBDL2 is regulated by TNF alpha and EGF, and plays an important role in facilitating keratinocyte migration. These results shed light on the interplay between TM ectodomain shedding and biological functions of keratinocytes, and further investigation is warranted.