J. Lynn Rutkowski

Neuregulin signaling through a PI3K/Akt/Bad pathway in Schwann cell survival.

beta-Neuregulin (betaNRG) is a potent Schwann cell survival factor that binds to and activates a heterodimeric ErbB2/ErbB3 receptor complex. We found that NRG receptor signaling rapidly activated phosphoinositide 3-kinase (PI3K) in serum-starved Schwann cells, while PI3K inhibitors markedly exacerbated apoptosis and completely blocked NRG-mediated rescue. NRG also rapidly signaled the phosphorylation of mitogen-activated protein kinase (MAPK) and the serine/threonine kinase Akt. The activation of Akt and MAPK in parallel pathways downstream from PI3K resulted in the phosphorylation of Bad at different serine residues. PI3K inhibitors that blocked NRG-mediated rescue also blocked the phosphorylation of Akt, MAPK, and Bad. However, selective inhibition of MEK-dependent Bad phosphorylation downstream from PI3K had no effect on NRG-mediated survival. Conversely, ectopic expression of wild-type Akt not only enhanced Bad phosphorylation but also enhanced autocrine- and NRG-mediated Schwann cell survival. Taken together, these results demonstrate that NRG receptor signaling through a PI3K/Akt/Bad pathway functions in Schwann cell survival.

RalGDS Functions in Ras- and cAMP-mediated Growth Stimulation

Thyroid-stimulating hormone stimulates proliferation through both the cAMP-dependent protein kinase and Ras but not through Raf-1 and mitogen-activated and extracellular signal-related kinase kinase. We now report that thyroid-stimulating hormone represses mitogen-activated protein kinase activity and that microinjection of an effector domain mutant Ha-Ras protein, Ras(12V,37G), defective in Raf-1 binding and mitogen-activated protein kinase activation, stimulates DNA synthesis in quiescent and thyroid-stimulating hormone-treated thyrocytes. A yeast two-hybrid screen identified RalGDS as a Ras(12V,37G) binding protein and therefore a potential effector of Ras in these cells. Associations between Ras and RalGDS were observed in extracts prepared from thyroid cells. Microinjection of a mutant RalA(28N) protein thought to sequester RalGDS family members reduced DNA synthesis stimulated by Ras as well as cAMP-mediated DNA synthesis in two cell lines which respond to cAMP with mitogenesis. These results support the idea that RalGDS may be an effector of Ras in cAMP-mediated growth stimulation.

Signals for proinflammatory cytokine secretion by human Schwann cells

Wallerian degeneration is a post-traumatic process of the peripheral nervous system whereby damaged axons and their surrounding myelin sheaths are phagocytosed by infiltrating leukocytes. Our studies indicate that Schwann cells could initiate the process of Wallerian degeneration by releasing proinflammatory cytokines involved in leukocyte recruitment and differentiation including IL-1beta, MCP-1, IL-8 and IL-6. A comparison of the secretory pattern between nerve explants and cultured Schwann cells showed that each cytokine was differentially regulated by growth factor deprivation or axonal membrane fragments. Since Wallerian-like degeneration occurs in a wide variety of peripheral neuropathies, Schwann cell-mediated cytokine production may play an important role in many disease processes.

The Zinc Finger Transcription Factor Zif268/Egr-1 Is Essential for Schwann Cell Expression of the p75 NGF Receptor

Nerve injury alters the function of Schwann cells from quiescent, myelin forming cells to proliferating cells that facilitate nerve repair. The transcription factor, Zif268, may be involved in transmitting injury-related signals since its expression is rapidly induced by nerve transection in vivo and without intervening protein synthesis by injury-related signals in vitro. Expression of the low-affinity p75 nerve growth factor receptor (NGFRp75) by Schwann cells after nerve injury closely correlated with the zif268 expression profile, and Zif268 transactivated the NGFRp75 promoter in transient transfection assays. Conversely, the NGFRp75 gene was not expressed when Zif268 protein was depleted by stable transfection of antisense cDNA. Moreover, nuclear proteins corresponding to Zif268 bound to the NGFRp75 promoter by Southwestern blotting, indicating that a direct interaction of Zif268 with the NGFR gene is required for its expression in Schwann cells.

Germline mutations in NF1 patients with malignancies.

We have analyzed 98.5% of the coding region of the NF1 gene at the cDNA level in seven NF1 patients who developed malignant peripheral nerve sheath tumors. Seven germline mutations were detected in six individuals: a 6‐bp in‐frame deletion in exon 28, a splice acceptor mutation in intron 31 resulting in a premature stop of translation, a missense mutation in exon 38, and three total NF1 gene deletions. In one of the patients with a total NF1 gene deletion, a missense mutation in exon 16 on the other NF1 allele was detected. These data indicate that NF1 patients developing malignant neoplasms can have any type of NF1 germline mutation such as a total gene deletion, a frameshift mutation, an in‐frame deletion, or a missense mutation. We conclude that in our series no specific type of NF1 germline mutation was found in NF1 individuals with malignancies, but that large NF1 gene deletions were more frequently found in this group than reported for the general population of NF1 individuals. Genes Chromosomes Cancer 26:376–380, 1999.

Reciprocal Id expression and myelin gene regulation in Schwann cells

Id proteins are thought to act as dominant negative antagonists of basic helix-loop-helix (bHLH) transcription factors that direct differentiation in various cell types. We found that Schwann cells express all four Id-family genes and that their transcript levels were reciprocally regulated in pairs during nerve maturation in vivo and cAMP-mediated differentiation in vitro. The rapid induction as part of the early response to axonal membranes and cytokines suggested that Id3 is involved in myelin gene repression. An inverse relationship between Id1/3 and myelin P0 expression was consistent with a role for these two Id proteins as inhibitors of differentiation, and Id1/3 proteins strongly repressed myelin gene promoter activity. Nuclear factors isolated from Schwann cells and intact sciatic nerves were found to bind three different HLH recognition sequences (E boxes) in the proximal region of the P0 promoter, and production of these DNA binding complexes was altered during differentiation. These data support the concept that Id proteins regulate myelin gene expression by controlling the formation of specific bHLH DNA binding complexes with different E-box preferences.

Sources of Human Schwann Cells and the Influence of Donor Age

We evaluated several tissues as possible sources for culturing human Schwann cells. The average cell yield (total cell number/mg of nerve fascicle) obtained from adult autopsy cases and transplant organ donors was similar (2 x 10(4) and 2.9 x 10(4), respectively), but significantly higher yields were obtained from dorsal roots of pediatric patients undergoing selective dorsal rhizotomy (6.1 x 10(4)). Fresh tissue was not essential since cells isolated from 0 to 20 h postmortem were equally viable. However, we found evidence that donor age affects the intrinsic growth rate of Schwann cells and perineurial fibroblasts in culture.

Schwann cell-conditioned medium inhibits angiogenesis in vitro and in vivo

BACKGROUND Neuroblastomas are biologically heterogeneous tumors that consist of two main cell populations: neuroblastic/ganglionic cells and Schwann cells. The amount of Schwannian stroma strongly impacts prognosis. Low tumor vascularity, localized stage, and favorable outcome are associated with tumors that are Schwannian stroma-rich/stroma-dominant. PROCEDURE To investigate if Schwann cells play a role in inhibiting angiogenesis in neuroblastoma tumors, we examined the ability of human Schwann cell-conditioned medium to affect bFGF- and VEGF-induced endothelial cell proliferation and migration, and in vivo angiogenesis. RESULTS Schwann cell-conditioned medium significantly inhibited bFGF- and VEGF-induced endothelial cell proliferation and migration. This effect appears to be specific for endothelial cells as smooth muscle cell and fibroblast proliferation were not inhibited by this medium. Schwann cell-conditioned medium also inhibited in vivo angiogenesis in rat corneal assays. CONCLUSIONS Schwann cells produce a potent inhibitor(s) of angiogenesis that may be responsible for the low level of vascularity and more benign clinical behavior of Schwannian stroma-rich/stroma-dominant neuroblastoma tumors. Studies to identify the inhibitor(s) are ongoing.

Lack of NF1 expression in a sporadic schwannoma from a patient without neurofibromatosis

SummaryThe neurofibromatosis type 1 (NF1) gene encodes a tumor suppressor protein, neurofibromin, which is expressed at high levels in Schwann cells and other adult tissues. Loss ofNF1 expression has been reported in Schwann cell tumors (neurofibrosarcomas) from patients with NF1 and its loss is associated with increased proliferation of these cells. In this report, we describe downregulation ofNF1 expression in a single spinal schwannoma from an individual without clinical features of neurofibromatosis type 1 or 2. Barely detectable expression ofNF1 RNA was found in this tumor byin situ hybridization using anNF1-specific riboprobe as well as by Northern blot and reverse-transcribed (RT)-PCR analysis. In Schwann cells cultured from this schwannoma, abundant expression ofNF1 RNA could be detected by Northern blot and RT-PCR analysis. These results suggest that, in some tumors, expression ofNF1 may be downregulated by factors produced within the tumor and may represent a novel mechanism for inactivating these growth suppressing genes and allowing for increased cell proliferation in tumors.