Takanori Kumano

HMG-CoA reductase inhibitor-induced L6 myoblast cell death: involvement of the phosphatidylinositol 3-kinase pathway

Our previous studies have shown that the HMG‐CoA reductase inhibitor (HCRI) causes rhabdomyolysis and electrical myotonia in rabbits and also kills L6 myoblasts in culture. In the present study, we analyzed the intracellular signal transduction pathway of HCRI‐induced cell death using L6 myoblasts as a model system. Here, we report that simvastatin, a lipophilic HCRI, efficiently inhibited isoprenylation of Ras proteins and therefore induced translocation of a significant part of Ras proteins from the membrane fraction into the cytosolic fraction within 10 min. With this translocation, PI 3‐kinase activity of the Ras‐bound form both in total and in the membrane fraction was also decreased profoundly. Furthermore, various PI 3‐kinase inhibitors also caused cell death with morphological changes similar to those caused by simvastatin. These results might represent the molecular events of HCRI‐induced cell death, and suggest the significance of PI 3‐kinase activity of the Ras‐bound form in the maintenance of cell viability.

INVOLVEMENT OF TYROSINE PHOSPHORYLATION IN HMG-COA REDUCTASE INHIBITOR-INDUCED CELL DEATH IN L6 MYOBLASTS

Our previous studies have shown that the HMG‐CoA reductase (HCR) inhibitor (HCRI), simvastatin, causes myopathy in rabbits and kills L6 myoblasts. The present study was designed to elucidate the molecular mechanism of HCRI‐induced cell death. We have demonstrated that simvastatin induces the tyrosine phosphorylation of several cellular proteins within 10 min. These phosphorylations were followed by apoptosis, as evidenced by the occurrence of internucleosomal DNA fragmentation and by morphological changes detected with Nomarski optics. Simvastatin‐induced cell death was prevented by tyrosine kinase inhibitors. The MTT assay revealed that the addition of mevalonic acid into the culture medium partially inhibited simvastatin‐induced cell death. Thus, these results suggested that protein tyrosine phosphorylation might play an important role in the intracellular signal transduction pathway mediating the HCRI‐induced death of myoblasts.

Role of tyrosine phosphorylation of phospholipase C γ1 in the signaling pathway of HMG-CoA reductase inhibitor-induced cell death of L6 myoblasts

Our previous studies have shown that the HMG‐CoA reductase (HCR) inhibitor (HCRI), simvastatin, kills L6 myoblasts by involving Ca2+ mobilization from the Ca2+ pool in the cells but not by influx from extracellular space. More recently, we found that HCRI induced tyrosine phosphorylation of several cellular proteins, followed by apoptotic cell death of L6 myoblasts. The present study was aimed to elucidate the molecular target(s) of these tyrosine phosphorylations induced by HCRI and demonstrated that simvastatin induces tyrosine phosphorylation of phospholipase C (PLC) γ1. This tyrosine phosphorylation of PLC‐γ1 caused the increment of the intracellular inositol triphosphate (IP3) levels in L6 myoblasts. Pretreatment of the cells with herbimycin A, a specific inhibitor of protein tyrosine kinase, inhibited a simvastatin‐induced increase in IP3 level in the cells as well as tyrosine phosphorylation of PLC‐γ1. Interestingly, pretreatment of the cells with U‐73122, a specific inhibitor of PLC, prevented simvastatin‐induced cell death. Thus, these results strongly suggest that simvastatin‐induced tyrosine phosphorylation of PLC‐γ1 plays, at least in part, an important role for the development of simvastatin‐induced cell death.

HMG-CoA reductase inhibitor induces a transient activation of high affinity nerve growth factor receptor, Trk, and morphological differentiation with fatal outcome in PC12 cells

The present study was aimed at investigating the possible toxicity of simvastatin on a neuronal cell line, PC12 cells. Simvastatin clearly induced a transient morphological differentiation as evidenced by the occurrence of neurite outgrowth with a transient activation of the high affinity nerve growth factor receptor, Trk, but died at 36 h after its addition. Tyrosine autophosphorylation of the Trk protein also disappeared at 36 h after addition. During the morphological differentiation, NGF mRNA expression was upregulated transiently and returned to the basal level at 36 h after addition of simvastatin. These results suggest that simvastatin is neurotoxic and PC12 cells elicited a protective response, involving a transient activation of a Trk-mediated intracellular signal transduction pathway by an autocrine secretion of NGF, although these responses did not persist against pro-apoptotic signals and resulted in an apoptosis of the PC12 cells.

Tyrosine phosphorylation of the catalytic subunit p110 of phosphatidylinositol‐3 kinase induced by HMG‐CoA reductase inhibitor inhibits its kinase activity in L6 myoblasts

Previous studies from this laboratory have shown that 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitor (HCRI) causes apoptotic cell death of a muscle cell‐derived cell line, L6 myoblasts, by involving the phosphatidylinositol‐3 (PI‐3) kinase pathway and tyrosine phosphorylation of several cellular proteins, although the relationship between PI‐3 kinase pathway and tyrosine phosphorylation responses remained to be elucidated. Here, we show that HCRI induces tyrosine phosphorylation of catalytic subunit p110 of PI‐3 kinase as early as 5 min after addition of HCRI into culture medium. We could not detect the tyrosine phosphorylation of the regulatory subunit p85 of PI‐3 kinase under the present experimental conditions. Concomitantly, the kinase activity toward PI in p110 immunoprecipitates was decreased with a similar time course. Furthermore, both herbimycin A and genistein, potent inhibitors of tyrosine kinase activity, inhibited HCRI‐induced inhibition of PI‐3 kinase activity as well as HCRI‐induced apoptotic cell death. Once the catalytic subunit p110 becomes tyrosine‐phosphorylated, the regulatory subunit p85 appears to be dissociated from the catalytic subunit, because we observed a decreasing amount of p85 regulatory subunits in p110 immunoprecipitates in response to HCRI treatment. These results strongly suggest the novel function of tyrosine phosphorylation of catalytic subunit p110 of PI‐3 kinase in the regulation of its kinase activity. The tyrosine phosphorylation of these catalytic subunits may play an important role in the intracellular signal transduction of apoptotic cell death. To our knowledge, this is the first report that tyrosine phosphorylation of p110 catalytic subunit acts as a negative regulator of its kinase activity.

Improvement of apraxia of eyelid opening by wearing goggles

Apraxia of eyelid opening (ALO) is a non-paralytic motor abnormality characterised by difficulty in initiating the act of eyelid opening without blepharospasm. We found that wearing goggles improved the difficulty of opening eyes in two patients with ALO with parkinsonism. Wearing goggles is a simple method for improving daily life in patients with ALO.

Chorea-acanthocytosis with polyclonal antibodies to ganglioside GM1

A patient with chorea-acanthocytosis presenting with axonal neuropathy showed an elevation in IgM polyclonal antibodies to the GM1 ganglioside, which were estimated by enzyme-linked immunosorbent assay and complement-mediated liposome immune lysis assay (LILA). This is the first demonstration of such antibodies in chorea-acanthocytosis. Anti-GM1 antibodies might have directly caused the axonal neuropathy by binding to GM1 or cross-reactive antigens in the nerves.

Muscle MRI findings of HTLV-1-associated myelopathy

We report here the muscle MRI findings in two patients with human T-cell lymphotropic virus type I-associated myelopathy (HAM). It is known that thigh muscles are vulnerable in HAM patients, but detailed information about the affected muscles has not been available. Muscle MRI findings of these patients showed that thigh muscles, especially adductor magnus, and semimembranosus muscles were severely affected, but lower leg muscles were comparatively preserved. In these affected muscles, neurogenic changes were observed by EMG. We concluded that muscle MRI is very useful to estimate the affected muscles in HAM patients.