u-0126 and bryostatin-1

In mammals, degeneration of peripheral auditory neurons constitutes one of the main causes of sensorineural hearing loss. Unfortunately, to date, pharmacological interventions aimed at counteracting this condition have not presented complete effectiveness in protecting the integrity of cochlear neural elements. In this context, the protein kinase C (PKC) family of enzymes are important signalling molecules that play a role in preventing neurodegeneration after nervous system injury. The present study demonstrates, for the first time, that the PKC signalling pathway is directly neurotrophic to axotomised spiral ganglion neurons (SGNs). We found that PKCbetaI was strictly expressed by postnatal and adult SGNs both in situ and in vitro. In cultures of SGNs, we observed that activators of PKC, such as phorbol esters and bryostatin 1, induced neuronal survival and neurite regrowth in a manner dependent on the activation of PKCbetaI. The neuroprotective effects of PKC activators were suppressed by pre-treatment with LY294002 (a PI3K inhibitor) and with U0126 (a MEK inhibitor), indicating that PKC activators promote the survival and neurite outgrowth of SGNs by both PI3K/Akt and MEK/ERK-dependent mechanisms. In addition, whereas combining the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) was shown to provide only an additive effect on SGN survival, the interaction between PKC and neurotrophin signalling gave rise to a synergistic increase in SGN survival. Taken together, the data indicate that PKCbetaI activation represents a key factor for the protection of the integrity of neural elements in the cochlea.

Topics: Animals; Antineoplastic Agents; Axotomy; Brain-Derived Neurotrophic Factor; Bryostatins; Butadienes; Cell Survival; Cells, Cultured; Chromones; Enzyme Activation; Enzyme Inhibitors; Isoenzymes; Macrolides; Morpholines; Nerve Growth Factors; Nerve Regeneration; Neurons; Neuroprotective Agents; Neurotrophin 3; Nitriles; Protein Kinase C; Protein Kinase C beta; Rats; Rats, Wistar; Spiral Ganglion; Tetradecanoylphorbol Acetate

Cotreatment with a minimally toxic concentration of the protein kinase C (PKC) activator (and down-regulator) bryostatin 1 (BRY) induced a marked increase in mitochondrial dysfunction and apoptosis in U937 monocytic leukemia cells exposed to the proteasome inhibitor lactacystin (LC). This effect was blocked by cycloheximide, but not by alpha-amanitin or actinomycin D. Qualitatively similar interactions were observed with other PKC activators (eg, phorbol 12-myristate 13-acetate and mezerein), but not phospholipase C, which does not down-regulate the enzyme. These events were examined in relationship to functional alterations in stress (eg, SAPK, JNK) and survival (eg, MAPK, ERK) signaling pathways. The observations that LC/BRY treatment failed to trigger JNK activation and that cell death was unaffected by a dominant-interfering form of c-JUN (TAM67) or by pretreatment with either curcumin or the p38/RK inhibitor, SB203580, suggested that the SAPK pathway was not involved in potentiation of apoptosis. In marked contrast, perturbations in the PKC/Raf/MAPK pathway played an integral role in LC/BRY-mediated cell death based on evidence that pretreatment of cells with bisindolylmaleimide I, a selective PKC inhibitor, or geldanamycin, a benzoquinone ansamycin, which destabilizes and depletes Raf-1, markedly suppressed apoptosis. Furthermore, ERK phosphorylation was substantially prolonged in LC/BRY-treated cells compared to those exposed to BRY alone, and pretreatment with the highly specific MEK inhibitors, PD98059, U0126, and SL327, opposed ERK activation while protecting cells from LC/BRY-induced lethality. Together, these findings suggest a role for activation and/or dysregulation of the PKC/MAPK cascade in modulation of leukemic cell apoptosis following exposure to the proteasome inhibitor LC. (Blood. 2001;97:2105-2114)

Topics: Acetylcysteine; Amanitins; Aminoacetonitrile; Apoptosis; Benzoquinones; Bryostatins; Butadienes; Curcumin; Cysteine Endopeptidases; Dactinomycin; Diterpenes; Drug Synergism; Enzyme Activation; Flavonoids; Humans; Imidazoles; Indoles; JNK Mitogen-Activated Protein Kinases; Lactams, Macrocyclic; Lactones; Macrolides; Maleimides; MAP Kinase Kinase 4; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Neoplasm Proteins; Nitriles; Nucleic Acid Synthesis Inhibitors; p38 Mitogen-Activated Protein Kinases; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Kinase C; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-raf; Pyridines; Quinones; Terpenes; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Type C Phospholipases; U937 Cells; Ubiquitins