BackgroundTransforming growth factor-β (TGF-β) and matrix metalloproteinases (MMPs) are the multifunctional factors during diverse physiological and pathological processes including development, wound healing, proliferation, and cancer metastasis. Both TGF-β and MMPs have been shown to play crucial roles in brain pathological changes. Thus, we investigated the molecular mechanisms underlying TGF-β1-induced MMP-9 expression in brain astrocytes.MethodsRat brain astrocytes (RBA-1) were used. MMP-9 expression was analyzed by gelatin zymography and RT-PCR. The involvement of signaling molecules including MAPKs and NF-κB in the responses was investigated using pharmacological inhibitors and dominant negative mutants, determined by western blot and gene promoter assay. The functional activity of MMP-9 was evaluated by cell migration assay.ResultsHere we report that TGF-β1 induces MMP-9 expression and enzymatic activity via a TGF-β receptor-activated reactive oxygen species (ROS)-dependent signaling pathway. ROS production leads to activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun-N-terminal kinase (JNK) and then activation of the NF-κB transcription factor. Activated NF-κB turns on transcription of the MMP-9 gene. The rat MMP-9 promoter, containing a NF-κB cis-binding site, was identified as a crucial domain linking to TGF-β1 action.ConclusionsCollectively, in RBA-1 cells, activation of ERK1/2- and JNK-NF-κB cascades by a ROS-dependent manner is essential for MMP-9 up-regulation/activation and cell migration induced by TGF-β1. These findings indicate a new regulatory pathway of TGF-β1 in regulating expression of MMP-9 in brain astrocytes, which is involved in physiological and pathological tissue remodeling of central nervous system.
Although kinesins are known to transport neuronal proteins, it is not known what role they play in the targeting of their cargos to specific subcellular compartments in neurons. Here we present evidence that the K ؉ channel Kv4.2, which is a major regulator of dendritic excitability, is transported to dendrites by the kinesin isoform Kif17. We show that a dominant negative construct against Kif17 dramatically inhibits localization to dendrites of both introduced and endogenous Kv4. The electrophysiological properties of neurons arise from an intricate spatial distribution of ion channels and receptors on the plasma membranes of these cells. The complexity of this distribution is illustrated by experiments showing that nearly 500 distinct proteins reside at the postsynaptic density (1), each of which must be regulated at over 10,000 postsynaptic sites in a typical cortical pyramidal neuron (2). The fidelity of this distribution is critical, since mislocalization of ion channels and receptors can lead to the breakdown of signal transduction cascades that are essential for physiological functioning of neurons (3). These and many other observations suggest that neurons can create and maintain complex subcellular distributions of proteins. However, little is known about how this is accomplished at the molecular level.Progress toward elucidating the molecular mechanisms underlying subcellular localization of neuronal proteins has been made by identifying targeting motifs within the primary structure of transmembrane proteins that are localized in distinct subcellular compartments (4 -9). The voltage-gated K ϩ channel Kv4.2 has served as a model for dendritic targeting (10). It is a major regulator of excitability in the dendrites of hippocampal pyramidal cells in the CA1 region, and its presence in dendrites but not axons is thought to account for the propagation of action potentials exclusively in the latter compartment (11). We previously identified a 16-amino acid dileucine-containing motif, located in the C terminus of Kv4.2 (10), that is both necessary for dendritic targeting of the channel and sufficient to induce nonspecifically localized transmembrane proteins to target to dendrites. Recent experiments suggest that some transmembrane proteins are localized in dendrites as a result of directed vesicular transport (12) and that transport of proteins from the cell body to dendrites is mediated by kinesins (13,14). In light of these findings, we sought to identify the kinesin isoform responsible for transporting Kv4.2 and to examine a possible role for this kinesin in dendritic targeting of the channel. By screening a series of dominant negative constructs directed against specific kinesins, we identified Kif17 as being necessary for localization of Kv4.2. We established that Kif17 and Kv4.2 probably interact by showing that native forms of the two proteins colocalize in dissociated neurons and coimmunoprecipitate from brain lysate. Introduced forms of the two proteins coimmunoprecipitate when coexpressed in COS c...
Here we present evidence that the kinesin, Kif5B, is involved in the transportation and axonal targeting of Kv1 channels. We show that a dominant negative variant of Kif5B specifically blocks localization to the axon of expressed, tagged versions of Kv1.3 in cultured cortical slices. In addition, the dominant negative variant of Kif5B blocks axonal localization of endogenous Kv1.1, Kv1.2, and Kv1.4 in cortical neurons in dissociated cultures. We also found evidence that Kif5B interacts with Kv1 channels. Endogenous Kv1.2 colocalized with Kif5B in cortical neurons and coimmunoprecipitated with Kif5B from brain lysate. The T1 domain of Shaker K(+) channels has been shown to play a critical role in targeting the channel to the axon. We have three pieces of evidence to suggest that the T1 domain also mediates interaction between Kv1 channels and Kif5B: Addition of the T1 domain to a heterologous protein, TfR, is sufficient to cause the resulting fusion protein, TfRT1, to colocalize with Kif5B. Also, the T1 domain is necessary for interaction of Kv1.3 with Kif5B in a coimmunoprecipitation assay. Finally, dominant negative variants of Kif5B block axonal targeting of TfRT1, but have no effect on dendritic localization of TfR. Together these data suggest a model where Kif5B interacts with Kv1 channels either directly or indirectly via the T1 domain, causing the channels to be transported to axons.
Shaker K+ channels play an important role in modulating electrical excitability of axons. Recent work has demonstrated that the T1 tetramerization domain of Kv1.2 is both necessary and sufficient for targeting of the channel to the axonal surface [Gu, C., Jan, Y.N. & Jan, L.Y. (2003) Science,301, 646-649]. Here we use a related channel, Kv1.3, as a model to investigate cellular mechanisms that mediate axonal targeting. We show that the T1 domain of Kv1.3 is necessary and sufficient to mediate targeting of the channel to the axonal surface in pyramidal neurons in slices of cortex from neonatal rat. The T1 domain is also sufficient to cause preferential axonal localization of intracellular protein, which indicates that the domain probably does not work through compartment-specific endocytosis or compartment-specific vesicle docking. To determine whether the T1 domain mediates axonal trafficking of transport vesicles, we compared the trafficking of vesicles containing green fluorescent protein-labelled transferrin receptor with those containing the same protein fused with the T1 domain in living cortical neurons. Vesicles containing the wild-type transferrin receptor did not traffic to the axon, in accord with previously published results; however, those containing the transferrin receptor fused to T1 did traffic to the axon. These results are consistent with the T1 domain of Kv1.3 mediating axonal targeting by causing transport vesicles to traffic to axons and they represent the first evidence that such a mechanism might underlie axonal targeting.
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