Through their ability to degrade the extracellular matrix, proteases mediate cancer cell invasion and metastasis. Paradoxically, some serine protease inhibitors (serpins) are often overexpressed in human tumors. Using computational analysis, we found that the RNA level of protease nexin-1 (PN-1), a serpin that blocks numerous proteases activity, is significantly elevated in estrogen receptor-A-negative and in high-grade breast cancer. The in silico approach was complemented by mechanistic studies on two mammary cancer cell lines, the PN-1-negative 168FARN cells and the PN-1-positive 4T1 cells, both of which form primary mammary tumors, but only 4T1 tumors are able to metastasize to the lungs. We show that treatment of 168FARN cells with PN-1 stimulates extracellular signal-regulated kinase activation via low-density lipoprotein receptor-related protein-1 (LRP-1) binding, resulting in increased matrix metalloproteinase (MMP)-9 RNA, protein, and secreted activity. PN-1-silenced 4T1 cells express low MMP-9 levels. Moreover, injection of PN-1-silenced cells into mice did not affect 4T1 primary mammary tumor outgrowth; however, the tumors had impaired metastatic potential, which could be restored by reexpressing soluble MMP-9 in the PN-1-silenced 4T1 cells. Thus, using mammary tumor models, we describe a novel pathway whereby the serpin PN-1 by binding LRP-1 stimulates extracellular signal-regulated kinase signaling, MMP-9 expression, and metastatic spread of mammary tumors. Importantly, an analysis of 126 breast cancer patients revealed that those whose breast tumors had elevated PN-1 levels had a significantly higher probability to develop lung metastasis, but not metastasis to other sites, on relapse. These results suggest that PN-1 might become a prognostic marker in breast cancer.
Experiments in peripheral and central synapses reveal the regulatory mechanisms that enable trans-synaptic control of synapse development and maintenance by the L1-type CAM Neuroglian.
Glutamate is the main excitatory neurotransmitter of the central nervous system. Tissue-type plasminogen activator (tPA) is recognized as a modulator of glutamatergic neurotransmission. This attribute is exemplified by its ability to potentiate calcium signaling following activation of the glutamate-binding N-methyl-D-aspartate receptor (NMDAR). It has been hypothesized that tPA can directly cleave the NR1 subunit of the NMDAR and thereby potentiate NMDA-induced calcium influx. In contrast, here we show that this increase in NMDAR signaling requires tPA to be proteolytically active, but does not involve cleavage of the NR1 subunit or plasminogen. Rather, we demonstrate that enhancement of NMDAR function by tPA is mediated by a member of the Low-Density Lipoprotein Receptor (LDLR) family. Hence, this study proposes a novel functional relationship between tPA, the NMDAR, a LDLR and an unknown substrate which we suspect to be a serpin. Interestingly, whilst tPA alone failed to cleave NR1, cell-surface NMDARs did serve as an efficient and discrete proteolytic target for plasmin. Hence, plasmin and tPA can affect the NMDAR via distinct avenues. Altogether, we find that plasmin directly proteolyses the NMDAR whilst tPA functions as an indirect modulator of NMDA-induced events via LDLR engagement.
SUMMARY
The dimensions of axons and synaptic terminals determine cell-intrinsic properties of neurons; however, the cellular mechanisms selectively controlling establishment and maintenance of neuronal compartments remain poorly understood. Here, we show that two giant Drosophila Ankyrin2 isoforms, Ank2-L and Ank2-XL, and the MAP1B homolog Futsch form a membrane-associated microtubule-organizing complex that determines axonal diameter, supports axonal transport, and provides independent control of synaptic dimensions and stability. Ank2-L controls microtubule and synaptic stability upstream of Ank2-XL that selectively controls microtubule organization. Synergistically with Futsch, Ank2-XL provides three-dimensional microtubule organization and is required to establish appropriate synaptic dimensions and release properties. In axons, the Ank2-XL/Futsch complex establishes evenly spaced, grid-like microtubule organization and determines axonal diameter in the absence of neurofilaments. Reduced microtubule spacing limits anterograde transport velocities of mitochondria and synaptic vesicles. Our data identify control of microtubule architecture as a central mechanism to selectively control neuronal dimensions, functional properties, and connectivity.
Targeted domain-specific mutations and cell type–specific rescue experiments show that L1CAM/Neuroglian is required for axon–axon interactions and yield insights into the cellular mechanisms controlling establishment of the complex mushroom body architecture.
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