Abstract
Thrombospondin Type I Domain Containing 7A (THSD7A) is a novel protein that mediates endothelial cell migration in angiogenesis. Bioinformatic analysis predicted THSD7A to be a membrane protein containing eleven thrombospondin-type-I repeats (TSR), one CD36-binding domain, and one RGD motif. These features imply THSD7A may be involved in cell migration and cell-to-ECM interaction. From previous studies, we have learned THSD7A regulates human umbilical vein endothelial cells (HUVECs) migration in vitro and affects zebrafish intersegmental vessels (ISV) angiogenesis during its development in vivo. However, the underlying mechanism by which THSD7A affects vessel guidance and patterning remains unclear. Since THSD7A was predicted to be a membrane protein, we first performed immunocytochemistry analysis to examine the subcellular localization of endogenous THSD7A in HUVEC. We found that THSD7A could be detected at peripheral nucleus area, which suggests its de novo synthesis and processing in the endoplasmic reticulum (ER) and Golgi apparatus. In addition, THSD7A could also be found at focal adhesion sites that co-localized with avb3 integrin and paxillin at the extremities of actin cytoskeleton in HUVECs. Taken together, these findings implied THSD7A may be a focal adhesion protein that was transported to membrane through ER-Golgi. THSD7A has a RGD motif, which is well known to interact with integrins and to activate them. Upon integrin activation, a focal adhesion-associated adaptor protein called paxillin is known to play as a key role in regulating cell motility. Therefore, we hypothesized that THSD7A may also regulate cell motility through the integrin-paxillin pathway. We found that THSD7A can be observed at the leading front of directed migrating cells, and THSD7A was similarly distributed as paxillin in migrating cells. We then verified the physical interaction of THSD7A and paxillin by co-immunoprecipitation assay. Furthermore, THSD7A knockdown in HUVECs induced the formation of broad lamellipodia-like structures and actin condensation, which are similar to paxillin-deficient cells observed in other studies. Altogether, these findings suggested THSD7A is a new binding partner of paxillin and it may mediate cell migration through integrin-paxillin-associated cytoskeletal reorganization. In summary, our studies revealed THSD7A has a wide range of effects beyond cell migration on primary endothelial cells. Further analysis of THSD7A will shed light on physiological mechanisms of endothelial cells, and may provide new insights into relevant pathological disorders.