Studies On Nogo-B Fuctions On Fibulin-5 In Cell Motility

Cell motility does not happen autonomously; their commitment to adhesion, migration and invasion depends upon environmental cues or messages which exist especially in extracellular matrix (ECM). The Fibulin family is a component of them and comprises six members which vary in tissue distribution. Fibulin-5 was identified by two groups in an attempt to isolate novel regulators of vascular growth [Kowal et al,1999; Nakamura et al,1999]. This 66 KDa ECM protein composes of 448-amino acids, and has interesting structural features; it contains an N-terminal cbEGF (calcium-binding epidermal growth factor)-like domain with an Arg-Gly-Asp: cell-adhesion motif, five contiguous cbEGF-like domains and a C-terminal fibulin-type module. Fibulin-5 figures prominently in the formation and stabilization of basement membranes, elastic fibers, and loose connective tissues, where they interact with a variety of other extracellular matrix components, including fibronectin, laminin, nidogen, aggrecan, versican, endostatin, elastin and et[Kostka, Get al 2001; Nakamura, T.et al,2002; Yanagisawa, H.et al,2002]. In addition to its structural functions, evidence now implicates that FBLN-5 is a mediator of cell-matrix communication and is involved in regulating organogenesis, vasculogenesis, fibrogenesis, and tumorigenesis. More recently, the aberrant malignancies suggests that reduced FBLN-5 expression may enhance cancer formation and progression in humans. Despite these recent advances, questions regarding this important ECM protein involved in cell motility remain to be unanswered, particularly, what are the molecules that regulate FBLN-5 secretion. Though, several groups have previously identified many FBLN-5-binding proteins involved in some signal pathways, such as lysyl oxidase-like 1 pathway, WNT pathway, ERK pathway and ect. None of them functions on FBLN-5 secretion or intracellular transportation. So, novel FBLN-5-binding proteins regulating these processes are the keys to uncover the molecular mechanism of how FBLN-5 playing its role on cell motility.Interestingly, we found one member of the reticulon family, Nogo-B (also known as RTN4b/Nogo-B), as a novel FBLN-5 binding protein by yeast-two hybridization from cDNA library of drosophila (data not shown). Nogo is one of the four members of the reticulon (RTN) family and it has three major isoforms (Nogo-A, B, and C), which are generated through alternative splicing and promoter use. All these isoforms of Nogo share a 66-amino-acid-residue luminal/extracellular domain (Nogo-66), which inhibits axonal extension and fibroblast spreading and a common C-terminal domain but diverse in the length of their N-terminal domains. Recent studies of the expression of Nogo isoforms reveals that Nogo-B is widely distribute over different cell types and tissues, where it has functions differently from one to another. Nogo-B was previously recognized as only an Endoplasmic reticulum (ER)-located protein and shows enhanced axonal regeneration in the Central Neural System (CNS), as well as being involved in some neural diseases:such as schizophrenia, depression, bipolar frontal cortex, atherosclerotic plaques and Alzheimer's disease [Kim, J. et al,2003; Gabriela Novaka et al, 2006; Juan A. et al,2007; Kume H, et al,2009; Bor Luen et al,2007]. Moreover, it is also considered as an apoptosis-induced gene, but this is still under argument [Thomas Oertle et al, 2002]. However, recently, Nogo-B has been found on the cell surface with interaction with the other two members (Nogo-A and Nogo-C) of the family, and has been reported to play an important role in adhesion and migration of cells as well as be involved in some processes related to cell motility, for example, the remodelling of the vasculature and neointima formation [Acevedo, L. et al,2004; Jacek J. et al,2007; Kritz AB et al,2008]. This is such a coincidence that FBLN-5 and Nogo-B do the similar jobs in certain biologic processes, such as cell motility and vascular remodeling. And following these cues, we finally got our new discovery.In the present study, we were first time to identify Nogo-B, which previously regarded as an apoptosis-relative or neural cell growth-relative protein can enhance FBLN-5 secretion and in turn stimulate cell motility. We investigated how Nogo-B interacted with FBLN-5, and how the interaction in turn regulated the adhesion, migration, invasion and proliferation of Hela cells.Firstly, Nogo-B bound FBLN-5 both in cytoplasm and on membrane, which was verified from the results of co-IP and confocal. Instead of enlarging its expression, Nogo-B enhanced FBLN-5 secretion by transporting more FBLN-5 to cell membrane. And this was proved by analyzing the quantity of intracellular and extracellular FBLN-5 of different stable Hela cell lines overexpressing Nogo-B. In addition, Nogo-B stimulated Hela cells to form pseudopods. Both of them enhanced Hela cell motility including migration and invasion. These data provided a molecular explanation for how Nogo-B enhanced cell migration and invasion in vitro and in vivo. Interestingly, overexpressing Nogo-B alleviated the adhesion and proliferation of Hela cell. We suspected this may due to the competitive inhibition of extensive FBLN-5 in the ECM. Too many soluble FBLN-5 contended for less ligands on the cell surface, which broke the proper linker between cell and ECM and made cell uneasy to adhere. And adhesion is one principle step of cell proliferation.Using recombinant mutants of human fibulin-5 and Nogo-B expressed by 293T, we also had mapped the precisely domains of both molecules interacting with each other. The sixth cbEGF domain of FBLN-5 and the Nogo-66 domain of Nogo-B took the responsibility of interacting. Though previous studies also found these two domains may play a role in cell movement, respectively. They didn't link them together and discover FBLN-5 and Nogo-B had such synergy effects. So, not only were our results in good agreement with previous findings, but it also revealed the critical relation underlying these processes.In addition, we also found over-expressing Nogo-B stimulated Hela cell to form pseudopods accompanied by its accumulation in pseudopods. And this determined the local assignment of FBLN-5 because Nogo-B carried FBLN-5 to the tips of pseudopods, with which cell pull themselves ahead. These findings indicated that the Nogo-B may work as a molecular transporter that carried the mature FBLN-5 protein to their destination. We propose that Nogo-B binds FBLN-5 molecule in ER or Golgi and finally transports it to the cell surface where it is released. Thus, it is likely that the physical association of the Nogo-B and FBLN-5 is an important event during the FBLN-5 secretion.In summary, we have identified Nogo-B as a FBLN-5 binding protein that can enhance FBLN-5 secretion. We demonstrated that the Nogo-B carried FBLN-5 to the cell surface and drove it to the pseudopods. Moreover, up-regulation of Nogo-B stimulates the formation of pseudopod in Hela cells. We proposed that Nogo-B might function as a molecular transporter that determines where FBLN-5 was carried to, thereby regulating subsequent secretion of FBLN-5 and cell movement. Further studies should focus on the molecular mechanism of FBLN-5 expression and the maturation process of it. With all these problems solved, we will able to outline the whole signal pathway which alter cell motility by means of regulating FBLN-5.