Jakub M. Swiercz

Plexin-B1 Directly Interacts with PDZ-RhoGEF/LARG to Regulate RhoA and Growth Cone Morphology

Plexins are widely expressed transmembrane proteins that, in the nervous system, mediate repulsive signals of semaphorins. However, the molecular nature of plexin-mediated signal transduction remains poorly understood. Here, we demonstrate that plexin-B family members associate through their C termini with the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG. Activation of plexin-B1 by semaphorin 4D regulates PDZ-RhoGEF/LARG activity leading to RhoA activation. In addition, a dominant-negative form of PDZ-RhoGEF blocks semaphorin 4D-induced growth cone collapse in primary hippocampal neurons. Our study indicates that the interaction of mammalian plexin-B family members with the multidomain proteins PDZ-RhoGEF and LARG represents an essential molecular link between plexin-B and localized, Rho-mediated downstream signaling events which underly various plexin-mediated cellular phenomena including axonal growth cone collapse.

Plexin-B1/RhoGEF–mediated RhoA activation involves the receptor tyrosine kinase ErbB-2

Plexins are widely expressed transmembrane proteins that mediate the effects of semaphorins. The molecular mechanisms of plexin-mediated signal transduction are still rather unclear. Plexin-B1 has recently been shown to mediate activation of RhoA through a stable interaction with the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG. However, it is unclear how the activity of plexin-B1 and its downstream effectors is regulated by its ligand Sema4D. Here, we show that plexin-B family members stably associate with the receptor tyrosine kinase ErbB-2. Binding of Sema4D to plexin-B1 stimulates the intrinsic tyrosine kinase activity of ErbB-2, resulting in the phosphorylation of both plexin-B1 and ErbB-2. A dominant-negative form of ErbB-2 blocks Sema4D-induced RhoA activation as well as axonal growth cone collapse in primary hippocampal neurons. Our data indicate that ErbB-2 is an important component of the plexin-B receptor system and that ErbB-2–mediated phosphorylation of plexin-B1 is critically involved in Sema4D-induced RhoA activation, which underlies cellular phenomena downstream of plexin-B1, including axonal growth cone collapse.

ErbB-2 and Met Reciprocally Regulate Cellular Signaling via Plexin-B1

Sema4D-induced activation of plexin-B1 has been reported to evoke different and sometimes opposing cellular responses. The mechanisms underlying the versatility of plexin-B1-mediated effects are not clear. Plexin-B1 can associate with the receptor tyrosine kinases ErbB-2 and Met. Here we show that Sema4D-induced activation and inactivation of RhoA require ErbB-2 and Met, respectively. In breast carcinoma cells, Sema4D can have pro- and anti-migratory effects depending on the presence of ErbB-2 and Met, and the exchange of the two receptor tyrosine kinases is sufficient to convert the cellular response to Sema4D from pro- to anti-migratory and vice versa. This work identifies a novel mechanism by which plexin-mediated signaling can be regulated and explains how Sema4D can exert different biological activities through the differential association of its receptor with ErbB-2 and Met.

Sema3E–Plexin D1 signaling drives human cancer cell invasiveness and metastatic spreading in mice

Semaphorin 3E (Sema3E) is a secreted molecule implicated in axonal path finding and inhibition of developmental and postischemic angiogenesis. Sema3E is also highly expressed in metastatic cancer cells, but its mechanistic role in tumor progression was not understood. Here we show that expression of Sema3E and its receptor Plexin D1 correlates with the metastatic progression of human tumors. Consistent with the clinical data, knocking down endogenous expression of either Sema3E or Plexin D1 in human metastatic carcinoma cells hampered their metastatic potential when injected into mice, while tumor growth was not markedly affected. Conversely, overexpression of exogenous Sema3E in cancer cells increased their invasiveness, transendothelial migration, and metastatic spreading, although it inhibited tumor vessel formation, resulting in reduced tumor growth in mice. The proinvasive and metastatic activity of Sema3E in tumor cells was dependent on transactivation of the Plexin D1-associated ErbB2/Neu oncogenic kinase. In sum, Sema3E-Plexin D1 signaling in cancer cells is crucially implicated in their metastatic behavior and may therefore be a promising target for strategies aimed at blocking tumor metastasis.

A Molecular Basis of Analgesic Tolerance to Cannabinoids

Clinical usage of cannabinoids in chronic pain states is limited by their central side effects and the pharmacodynamic tolerance that sets in after repeated dosage. Analgesic tolerance to cannabinoids in vivo could be caused by agonist-induced downregulation and intracellular trafficking of cannabinoid receptors, but little is known about the molecular mechanisms involved. We show here that the type 1 cannabinoid receptor (CB1) interacts physically with G-protein-associated sorting protein 1 (GASP1), a protein that sorts receptors in lysosomal compartments destined for degradation. CB1–GASP1 interaction was observed to be required for agonist-induced downregulation of CB1 in spinal neurons ex vivo as well as in vivo. Importantly, uncoupling CB1 from GASP1 in mice in vivo abrogated tolerance toward cannabinoid-induced analgesia. These results suggest that GASP1 is a key regulator of the fate of CB1 after agonist exposure in the nervous system and critically determines analgesic tolerance to cannabinoids.

Plexin-B2, But Not Plexin-B1, Critically Modulates Neuronal Migration and Patterning of the Developing Nervous System In Vivo

Semaphorins and their receptors, plexins, have emerged as important cellular cues regulating key developmental processes. B-type plexins directly regulate the actin cytoskeleton in a variety of cell types. Recently, B-type plexins have been shown to be expressed in striking patterns in the nervous system over critical developmental windows. However, in contrast to the well characterized plexin-A family, the functional role of plexin-B proteins in neural development and organogenesis in vertebrates in vivo is not known. Here, we have elucidated the functional contribution of the two neuronally expressed plexin-B proteins, Plexin-B1 or Plexin-B2, toward the development of the peripheral nervous system and the CNS by generating and analyzing constitutive knock-out mice. The development of the nervous system was found to be normal in mice lacking Plexin-B1, whereas mice lacking Plexin-B2 demonstrated defects in closure of the neural tube and a conspicuous disorganization of the embryonic brain. After analyzing mutant mice, which bypassed neural tube defects, we observed a key requirement for Plexin-B2 in proliferation and migration of granule cell precursors in the developing dentate gyrus, olfactory bulb, and cerebellum. Furthermore, we identified semaphorin 4C as a high-affinity ligand for Plexin-B2 in binding and functional assays. Semaphorin 4C stimulated activation of ErbB-2 and RhoA via Plexin-B2 and enhanced proliferation and migration of granule cell precursors. Semaphorin 4C-induced proliferation of ventricular zone neuroblasts was abrogated in mice lacking Plexin-B2. These genetic and functional analyses reveal a key requirement for Plexin-B2, but not Plexin-B1, in patterning of the vertebrate nervous system in vivo.

Association of axon guidance factor Semaphorin 3A with poor outcome in pancreatic cancer

Neural alterations and aberrantly expressed nerve‐specific factors promoting tumor progression are known to contribute to pancreatic cancers extremely poor prognosis. Despite hints that axon guidance factor semaphorin 3A (SEMA3A) may function as a tumor inhibitor, its clinical importance and therapeutic potential have not yet been explored. The present study investigated the role of SEMA3A and its receptors—plexins A1–A4 (PLXNA1–A4) and neuropilin‐1 (NRP1)—in pancreatic cancer. QRT‐PCR and immunohistochemical analyses revealed overexpression of SEMA3A, NRP1 and PLXNA1 in metaplastic ducts, malignant cells and nerves of cancerous specimens, and showed that elevated levels of corresponding mRNA (6.8‐fold, 2.0‐fold and 1.5‐fold, respectively) clearly correlated with negative clinicopathological manifestations such as shorter survival (SEMA3A and PLXNA1) and a lesser degree of tumor differentiation (NRP1) in Stages I–III patients. High SEMA3A expression in pancreata of Stage IV M1 patients and in peritoneal metastases, and consequent functional studies indicated that poor clinical outcome might be related to the ability of SEMA3A to promote dissemination and invasiveness of pancreatic cancer cells through activation of multiple pathways involving Rac1, GSK3b or p42/p44 MAPK, but not E‐ to N‐cadherin switch, MMP‐9 or VEGF induction. Thus, this study is the first to quantify expression of the SEMA3A system in human malignancy and to show that overexpression of SEMA3A by nerves and transformed cells leads to a SEMA3A‐rich environment which may favor malignant activities of tumor cells. Furthermore, negative clinicopathological correlations suggest that SEMA3A might represent a novel intervention target but not a treatment option for pancreatic cancer patients.

ErbB-2 signals through Plexin-B1 to promote breast cancer metastasis

Diagnosis of metastatic breast cancer is associated with a very poor prognosis. New therapeutic targets are urgently needed, but their development is hampered by a lack of understanding of the mechanisms leading to tumor metastasis. Exemplifying this is the fact that the approximately 30% of all breast cancers overexpressing the receptor tyrosine kinase ErbB-2 are characterized by high metastatic potential and poor prognosis, but the signaling events downstream of ErbB-2 that drive cancer cell invasion and metastasis remain incompletely understood. Here we show that overexpression of ErbB-2 in human breast cancer cell lines leads to phosphorylation and activation of the semaphorin receptor Plexin-B1. This was required for ErbB-2-dependent activation of the pro-metastatic small GTPases RhoA and RhoC and promoted invasive behavior of human breast cancer cells. In a mouse model of ErbB-2-overexpressing breast cancer, ablation of the gene encoding Plexin-B1 strongly reduced the occurrence of metastases. Moreover, in human patients with ErbB-2-overexpressing breast cancer, low levels of Plexin-B1 expression correlated with good prognosis. Our data suggest that Plexin-B1 represents a new candidate therapeutic target for treating patients with ErbB-2-positive breast cancer.

Characterization of the expression of PDZ‐RhoGEF, LARG and Gα12/Gα13 proteins in the murine nervous system

Small GTPases of the Rho‐family, like Rho, Rac and Cdc42, are involved in neuronal morphogenesis by regulating growth cone morphology or dendritic spine formation. G‐proteins of the G12‐family, G12 and G13, couple G‐protein‐coupled receptors (GPCRs) to the activation of RhoA. Recently, two novel Rho‐specific guanine nucleotide exchange factors (RhoGEFs), PDZ‐RhoGEF and LARG, have been identified to interact with the activated α‐subunits of G12/G13 and are thus believed to mediate GPCR‐induced Rho activation. Although studies in neuronal cell lines have shown that G12/G13 and PDZ‐RhoGEF mediate GPCR‐induced neurite retraction, the role, as well as the expression of this signalling pathway, in intact brain has not been adequately studied. In the present study, we have characterized systematically the expression of Gα12, Gα13, PDZ‐RhoGEF and LARG in various murine tissues as well as their subcellular localization in the central and peripheral nervous systems. By performing immunohistochemistry, using polyclonal antibodies raised against the above proteins, we observed that Gα12, Gα13 and their RhoGEF‐effectors are distributed widely in the mammalian nervous system. Moreover, these proteins localize to distinct morphological compartments within neurons. While LARG and Gα12 were mainly found in somata of the neurons, PDZ‐RhoGEF and Gα13 were predominantly localized in the neuropil of central neurons. Interestingly, PDZ‐RhoGEF is a neural‐specific protein, whereas LARG is nearly ubiqoutous. Our data provide evidence that the G12/13–RhoGEF‐mediated pathway is present throughout the adult brain and may be involved in regulation of neuronal morphogenesis and function via GPCRs.

A functional role for semaphorin 4D/plexin B1 interactions in epithelial branching morphogenesis during organogenesis

Semaphorins and their receptors, plexins, carry out important functions during development and disease. In contrast to the well-characterized plexin A family, however, very little is known about the functional relevance of B-type plexins in organogenesis, particularly outside the nervous system. Here, we demonstrate that plexin B1 and its ligand Sema4d are selectively expressed in epithelial and mesenchymal compartments during key steps in the genesis of some organs. This selective expression suggests a role in epithelial-mesenchymal interactions. Importantly, using the developing metanephros as a model system, we have observed that endogenously expressed and exogenously supplemented Sema4d inhibits branching morphogenesis during early stages of development of the ureteric collecting duct system. Our results further suggest that the RhoA-ROCK pathway, which is activated downstream of plexin B1, mediates these inhibitory morphogenetic effects of Sema4d and suppresses branch-promoting signalling effectors of the plexin B1 signalling complex. Finally, mice that lack plexin B1 show early anomalies in kidney development in vivo. These results identify a novel function for plexin B1 as a negative regulator of branching morphogenesis during kidney development, and suggest that the Sema4d-plexin B1 ligand-receptor pair contributes to epithelial-mesenchymal interactions during organogenesis via modulation of RhoA signalling.