Masami Tsukamoto

Rho-kinase inhibition prevents the progression of diabetic nephropathy by downregulating hypoxia-inducible factor 1α

The small GTPase Rho and its effector Rho-kinase are involved in the pathogenesis of diabetic nephropathy. Accumulating evidence shows that hypoxia-inducible factor-1α (HIF-1α) is a key regulator of renal sclerosis under diabetic conditions. However, the interactions of Rho-kinase and HIF-1α in the development of renal dysfunction have not been defined. Here, we assessed whether Rho-kinase blockade attenuates HIF-1α induction and the subsequent fibrotic response using type 2 diabetic mice and cultured mesangial cells. Fasudil, a Rho-kinase inhibitor, reduced urinary albumin excretion, mesangial matrix expansion, and the expression of fibrotic mediators in db/db mice. Mechanistically, HIF-1α accumulation and the expression of its target genes that contribute to diabetic glomerulosclerosis were also prevented by fasudil in the renal cortex. In mesangial cells, Rho/Rho-kinase signaling was activated under hypoxic conditions. Further in vitro studies showed that pharmacological and genetic inhibition of Rho-kinase promoted proteasomal HIF-1α degradation, which subsequently suppressed HIF-1-dependent profibrotic gene expression by upregulation of prolyl hydroxylase 2. Thus, we found a previously unrecognized renoprotective mechanism for the effects of Rho-kinase inhibition and this could be a potential therapeutic target for the treatment of diabetic nephropathy.

Rho-kinase regulation of TNF-α-induced nuclear translocation of NF-κB RelA/p65 and M-CSF expression via p38 MAPK in mesangial cells

The small GTPase Rho and its downstream effector, Rho-associated coiled-coil containing protein kinase (Rho-kinase), regulate a number of cellular processes, including organization of the actin cytoskeleton, cell adhesion, and migration. While pharmacological inhibitors of Rho-kinase signaling are known to block renal inflammation, the molecular basis for this effect is unclear. Here, we provide evidence that proinflammatory TNF-α promotes mesangial expression of macrophage colony-stimulating factor (M-CSF), a key regulator for the growth and differentiation of mononuclear phagocytes, in a Rho-kinase-dependent manner. Consistent with this observation, TNF-α-mediated renal expression of M-CSF in insulin-resistant db/db mice was downregulated by Rho-kinase inhibition. Small interfering RNA-facilitated knockdown of Rho-kinase isoforms ROCK1 and ROCK2 indicated that both isoforms make comparable contributions to regulation of M-CSF expression in mesangial cells. From a mechanistic standpoint, Western blotting and EMSA showed that Rho-kinase and its downstream target p38 MAPK regulate nuclear translocation of NF-κB RelA/p65 and subsequent DNA binding activity, with no significant effects on IκBα degradation and RelA/p65 phosphorylation. Moreover, we showed that Rho-kinase-mediated cytoskeletal organization is required for the nuclear uptake of RelA/p65. Collectively, these findings identify Rho-kinase as a critical regulator of chemokine expression and macrophage proliferation.

Adenoviral expression of TDP‐43 and FUS genes and shRNAs for protein degradation pathways in rodent motoneurons in vitro and in vivo

Formation of cytoplasmic aggregates in neuronal and glial cells is one of the pathological hallmarks of amyotrophic lateral sclerosis (ALS). Mutations in two genes encoding transactivation response (TAR) DNA‐binding protein 43 (TDP‐43) and fused in sarcoma (FUS), both of which are main constituents of cytoplasmic aggregates, have been identified in patients with familial and sporadic ALS. Impairment of protein degradation machineries has also been recognized to participate in motoneuron degeneration in ALS. In the present study, we produced recombinant adenovirus vectors encoding wild type and mutant TDP‐43 and FUS, and those encoding short hairpin RNAs (shRNAs) for proteasome (PSMC1), autophagy (ATG5), and endosome (VPS24) systems to investigate whether the coupled gene transductions in motoneurons by these adenoviruses elicit ALS pathology. Cultured neurons, astrocytes and oligodendrocytes differentiated from adult rat neural stem cells and motoneurons derived from mouse embryonic stem cells were successfully infected with these adenoviruses showing cytoplasmic aggregate formation. When these adenoviruses were injected into the facial nerves of adult rats, exogenous TDP‐43 and FUS proteins were strongly expressed in facial motoneurons by a retrograde axonal transport of the adenoviruses. Co‐infections of adenovirus encoding shRNA for PSMC1, ATG5 or VPS24 with TDP‐43 or FUS adenovirus enhanced cytoplasmic aggregate formation in facial motoneurons, suggesting that impairment of protein degradation pathways accelerates formation of TDP‐43 and FUS‐positive aggregates in ALS.

Upregulation of galectin-3 in immortalized Schwann cells IFRS1 under diabetic conditions

A spontaneously immortalized adult Fischer rat Schwann cell line IFRS1 retains the characteristic features of normal Schwann cells, and can be a useful tool for the study of diabetic neuropathy. In the present study, we examined the effects of high glucose and 3-deoxyglucosone (3-DG) on the viability and the protein expression of advanced glycation endproducts (AGE)-binding proteins, such as galectin-3 (GAL-3) and receptor for AGE (RAGE) in IFRS1 cells. Exposure to 30mM of glucose or 0.2mM of 3-DG for 7 days failed to impair the IFRS1 cell viability, but significantly upregulated the expression of GAL-3. The same exposure tended to increase the expression of RAGE, but the changes were not significant. The high glucose-induced upregulation of GAL-3 was attenuated by cotreatment with 0.2mM of an anti-glycated agent aminoguanidine or 20nM of an anti-oxidant trans-resveratrol. In addition, treatment of IFRS1 cells with 1μg/ml of recombinant GAL-3 for 48h resulted in the upregulation of B-cell lymphoma 2 (Bcl-2) and the downregulation of 4-hydroxynonenal (4HNE). These findings suggest the involvement of GAL-3 in the glycation and oxidative stress under diabetic conditions and its cytoprotective role in Schwann cells.

Fasudil inhibits ER stress-induced VCAM-1 expression by modulating unfolded protein response in endothelial cells

The process of atherosclerosis is affected by interactions among numerous biological pathways. Accumulating evidence shows that endoplasmic reticulum (ER) stress plays a crucial role in the development of atherosclerosis. Rho-kinase is an effector of small GTP-binding protein Rho, and has been implicated as an atherogenic factor. Previous studies demonstrated that fasudil, a specific Rho-kinase inhibitor, exerts a cardioprotective effect by downregulating ER stress signaling. However, the molecular link between ER stress and Rho-kinase in endothelial cells has not been elucidated. In this study, we investigated the mechanisms by which fasudil regulates endothelial inflammation during ER stress. Tunicamycin, an established ER stress inducer, increased vascular cellular adhesion molecule (VCAM)-1 expression in endothelial cells. Intriguingly, fasudil inhibited VCAM-1 induction. From a mechanistic stand point, fasudil inhibited expression of activating transcription factor (ATF)4 and subsequent C/EBP homologous protein (CHOP) induction by tunicamycin. Furthermore, fasudil attenuated tunicamycin-induced phophorylation of p38MAPK that is crucial for the atherogenic response during ER stress. These findings indicate that Rho-kinase regulates ER stress-mediated VCAM-1 induction by ATF4- and p38MAPK-dependent signaling pathways. Rho-kinase inhibition by fasudil would be an important therapeutic approach against atherosclerosis, in particular, under conditions of ER stress.

Involvement of oxidative stress and impaired lysosomal degradation in amiodarone‐induced schwannopathy

Amiodarone hydrochloride (AMD), an anti‐arrhythmic agent, has been shown to cause peripheral neuropathy; however, its pathogenesis remains unknown. We examined the toxic effects of AMD on an immortalized adult rat Schwann cell line, IFRS1, and cocultures of IFRS1 cells and adult rat dorsal root ganglion neurons or nerve growth factor‐primed PC12 cells. Treatment with AMD (1, 5, and 10 μm) induced time‐ and dose‐dependent cell death, accumulation of phospholipids and neutral lipids, upregulation of the expression of gangliosides, and oxidative stress (increased nuclear factor E2‐related factor in nuclear extracts and reduced GSH/GSSG ratios) in IFRS1 cells. It also induced the upregulation of LC3‐II and p62 expression, with phosphorylation of p62, suggesting that deficient autolysosomal degradation is involved in AMD‐induced IFRS1 cell death. Furthermore, treatment of the cocultures with AMD induced detachment of IFRS1 cells from neurite networks in a time‐ and dose‐dependent manner. These findings suggest that AMD‐induced lysosomal storage accompanied by enhanced oxidative stress and impaired lysosomal degradation in Schwann cells might be a cause of demyelination in the peripheral nervous system.

Spontaneously Immortalized Adult Rodent Schwann Cells as Valuable Tools for the Study of Peripheral Nerve Degeneration and Regeneration

We have established spontaneously immortalized Schwann cell lines from normal adult mice and rats, as well as murine disease models. One of the normal mouse cell lines, IMS32, possesses some biological properties of mature Schwann cells and high proliferative activities. The IMS32 cells have been utilized to investigate the action mechanisms of various molecules involved in peripheral nerve regeneration [e.g., ciliary neurotrophic factor (CNTF), sonic hedgehog, and galectin-1], and the pathogenesis of diabetic neuropathy, particularly the polyol pathway hyperactivity. The cell lines derived from murine disease models (e.g., lysosomal storage diseases, Charcot-Marie-Tooth disease, and neurofibromatosis) retain genomic and biochemical abnormalities, sufficiently representing the pathological features of the mutant mice. A normal rat cell line, IFRS1, retains the characteristic features of mature Schwann cells and the fundamental ability to myelinate axons in coculture with adult rat DRG neurons and PC12 cells. These Schwann cell lines can be valuable tools for exploring neuron–Schwann cell interactions, the pathobiology of axonal degeneration and regeneration in the peripheral nervous system, and novel therapeutic approaches against neurological disorders in patients with relevant diseases.

Physiological and Pathological Roles of Aldose Reductase in Schwann Cells

Aldose reductase (AR), the first enzyme in the polyol pathway, is predominantly localized to Schwann cells in the peripheral nervous system (PNS). The exaggerated glucose flux into the pathway via AR in Schwann cells under diabetic conditions is thought to be a major contributing factor in the pathogenesis of diabetic neuropathy, and the restoring effects of AR inhibitors on the neurological symptoms of experimental diabetic animals and patients with diabetes have been investigated. In contrast, however, much less attention has been paid to the physiological functions of AR in the PNS and other tissues (i.e. osmoregulation, aldehyde detoxification, and steroid and catecholamine metabolism). In this paper, we focus on the functional significance of AR in Schwann cells under normal and diabetic conditions. A spontaneously immortalized adult mouse Schwann cell line IMS32 displays distinct Schwann cell phenotypes and high glucose (30 mM)-induced upregulation of AR expression and accumulation of sorbitol and fructose. This cell line can be a useful model to study the physiological and pathological roles of AR in the PNS, especially the interactions between the polyol pathway and other pathogenetic factors of diabetic neuropathy, and the functional redundancy of AR and other enzymes in aldehyde detoxification.