leupeptins and Pheochromocytoma

Rat pheochromocytoma (PC12) cells were treated with the proteasome inhibitor MG-132 and morphological changes were recorded. Initially, neuronal differentiation was induced but after 24 h signs of morphological deterioration became apparent. We performed nuclear staining, flow cytometry and WST-1 assay then analyzed signal transduction pathways involving Akt, p38 MAPK (Mitogen-Activated Protein Kinase), JNK (c-Jun N-terminal Kinase), c-Jun and caspase-3. Stress signaling via p38, JNK and c-Jun was active even after 24 h of MG-132 treatment, while the survival-mediating Akt phosphorylation declined and the executor of apoptosis (caspase-3) was activated by that time and apoptosis was also observable. We examined subcellular localization of stress signaling components, applied kinase inhibitors and dominant negative H-Ras mutant-expressing PC12 cells in order to decipher connections of stress-mediating pathways. Our results are suggestive of that treatment with the proteasome inhibitor MG-132 has a biphasic nature in PC12 cells. Initially, it induces neuronal differentiation but prolonged treatments lead to apoptosis.

Topics: Adrenal Gland Neoplasms; Animals; Apoptosis; Caspase 3; Enzyme Activation; JNK Mitogen-Activated Protein Kinases; Leupeptins; p38 Mitogen-Activated Protein Kinases; PC12 Cells; Pheochromocytoma; Proteasome Inhibitors; Proto-Oncogene Proteins c-akt; Rats

Basic fibroblast growth factor (FGF-2) is up-regulated in response to a nerve lesion and promotes axonal regeneration by activation of the tyrosine kinase receptor fibroblast growth factor receptor 1 (FGFR1). To determine the effects of elevated FGFR1 levels on neurite outgrowth, overexpression was combined with lysosomal inhibition of receptor degradation. In pheochromocytoma (PC12) cells, FGFR1 overexpression resulted in flattened morphology, increased neurite outgrowth and activation of extracellular signal-regulated kinase (ERK) and AKT. Degradation of FGFR1 was inhibited by the lysosomal inhibitor leupeptin and by the proteasomal inhibitor lactacystin. In rat primary adult neurons, FGFR1 overexpression enhanced FGF-2-induced axon growth which was further increased by co-treatment with leupeptin. Lysosomal inhibition of receptor degradation concomitant with ligand stimulation of neurons overexpressing FGFR1 provides new insight in tyrosine kinase receptor-mediated promotion of axon regeneration and demonstrates that adult sensory neurons express sub-optimal levels of tyrosine kinase receptors for neurotrophic factors.

Topics: Acetylcysteine; Animals; Blotting, Western; Cysteine Proteinase Inhibitors; Fibroblast Growth Factors; Ganglia, Spinal; Green Fluorescent Proteins; Leupeptins; Ligands; Lysosomes; Neurites; PC12 Cells; Pheochromocytoma; Rats; Receptor, Fibroblast Growth Factor, Type 1; Reverse Transcriptase Polymerase Chain Reaction; Sensory Receptor Cells; Signal Transduction

A tripeptide aldehyde protease inhibitor (Ac-Leu-Leu-Nle-al, ALLNal), among many other protease inhibitors, initiates neurite outgrowth in PC12h cells. The neuritogenesis induced by this inhibitor is different in some aspects from that induced by NGF; one or two long neurites are initiated from the cell body and differentiation is transient. The results provide evidence that the protease inhibitor and nerve growth factor elicit neurite initiation by different mechanisms and suggest the existence of a novel protease which modulates neurite initiation in PC12 cells. To identify the target protease, Leu-Leu-Leu-al was immobilized and used as a ligand for affinity chromatography of the protease. A protein with an apparent molecular weight of 33 kDa was isolated specifically from the cytoplasmic fraction of PC12 cells using the affinity column. The same protein was identified in the brains of 1-day postnatal rats, but the amount was much less in the brains of adult rats. Thus, we suggest that the 33-kDa protein regulates neurite initiation in nervous systems, possibly as a protease which degrades membrane proteins or the cytoskeletal framework.

Topics: Adrenal Gland Neoplasms; Animals; Axons; Chromatography, Affinity; Dose-Response Relationship, Drug; Endopeptidases; Leupeptins; Molecular Weight; Osmolar Concentration; Pheochromocytoma; Protease Inhibitors; Tumor Cells, Cultured

We report here the possible involvement of a new protease in neurite initiation by PC12h cells. Addition of a leupeptin analogue (Ac-Leu-Leu-Nle-al, ALLNal) to PC12h cells on culture plates coated with collagen type I caused de novo neurite outgrowth. Other protease inhibitors (Ac-Leu-Leu-Met-al, leupeptin, E64c, E64d, soybean trypsin inhibitor, hirudin, aprotinin, diisofluorophosphate, 6-aminocapric acid, and pepstatin A) could not mimic this neurite-initiating action. ALLNal induced the initiation of one or two long neurites from the cell body, and increased the cellular level of acetylcholinesterase to an extent similar to nerve growth factor (NGF). However, ALLNal-induced neuritogenesis is different from that induced by NGF, in which many neurites are induced from a single cell body. In addition, in contrast to neurons induced by NGF, which survive for a long time, ALLNal-induced differentiation was transient, and after 48 h percentage of cells bearing neurites started to decrease. After about 120 h exposure to ALLNal, neurites had mostly disappeared and the acetylcholinesterase activity level was not as great as that produced by NGF. These results provide evidence that ALLNal and NGF elicit neurite initiation by different mechanisms, and suggest the existence of a regulatory system of neuronal differentiation through specific protease-protease inhibitor interaction.

Topics: Acetylcholinesterase; Amino Acid Sequence; Animals; Axons; Bucladesine; Cell Adhesion; Chemical Phenomena; Chemistry, Physical; Collagen; Growth; Leupeptins; Molecular Sequence Data; Nerve Fibers; Nerve Growth Factors; Oligopeptides; Pheochromocytoma; Protease Inhibitors; Rats; Time Factors; Tumor Cells, Cultured