Seijiro Mori

Platelet-derived Growth Factor Activates p38 Mitogen-activated Protein Kinase through a Ras-dependent Pathway That Is Important for Actin Reorganization and Cell Migration

Members of the mitogen activated protein (MAP) kinase family, extracellular signal-regulated kinase, stress-activated protein kinase-1/c-Jun NH2-terminal kinase, and p38, are central elements that transduce the signal generated by growth factors, cytokines, and stressing agents. It is well known that the platelet-derived growth factor (PDGF) activates extracellular signal-regulated kinase, which leads to cellular mitogenic response. On the other hand, the role of the other MAP kinases in mediating the cellular function of PDGF remains unclear. In the present study, we have investigated the functional role of the other MAP kinases in PDGF-mediated cellular responses. We show that ligand stimulation of PDGF receptors leads to the activation of p38 but not stress-activated protein kinase-1/c-Jun NH2-terminal kinase. Experiments using a specific inhibitor of p38, SB203580, show that the activation of p38 is required for PDGF-induced cell motility responses such as cell migration and actin reorganization but not required for PDGF-stimulated DNA synthesis. Analyses of tyrosine residue-mutated PDGF receptors show that Src homology 2 domain-containing proteins including Src family kinases, phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, the Src homology 2 domain-containing phosphatase SHP-2, phospholipase C-γ, and Crk do not play a major role in mediating the PDGF-induced activation of p38. Finally, the expression of dominant-negative Ras but not dominant-negative Rac inhibited p38 activation by PDGF, suggesting that Ras is a potent mediator in the p38 activation pathway downstream of PDGF receptors. Taken together, our present study proposes the existence of a Ras-dependent pathway for the activation of p38, which is important for cell motility responses elicited by PDGF stimulation.

Mice lacking Smad3 are protected against streptozotocin-induced diabetic glomerulopathy

Abstract Transforming growth factor-β (TGF-β) has been implicated in the development of diabetic glomerulopathy. In order to evaluate a role of Smad3, one of the major signaling molecules downstream of TGF-β, in the pathogenesis of diabetic glomerulopathy, Smad3-null mice were made diabetic with streptozotocin injection and analyzed 4 weeks after induction of diabetes. Electron microscopy revealed that the thickness of glomerular basement membrane (GBM) in wild-type diabetic mice was significantly higher than that in non-diabetic mice, whereas no appreciable GBM thickening was found in Smad3-null diabetic mice. Urinary albumin excretion was dramatically increased in wild-type diabetic mice, whereas Smad3-null diabetic mice did not show any overt albuminuria. Northern blotting revealed that mRNA levels of fibronectin and α3 chain of type IV collagen (α3Col4) in renal cortex of wild-type diabetic mice were approximately twice as much as those of non-diabetic mice, whereas their mRNA levels were not increased in Smad3-null diabetic mice. Real-time polymerase chain reaction (PCR) also confirmed diabetes-induced upregulation of fibronectin and α3Col4 in glomeruli of wild-type mice. Glomerular expression of TGF-β1, as assessed by real-time PCR, was enhanced to a similar degree in wild-type and smad3-null diabetic mice, indicating that the observed differences between wild-type and Smad3-null mice are not attributable to difference in the expression of TGF-β1. These data clearly demonstrate a critical role of Smad3 in the early phase of diabetic glomerulopathy. This may be due at least partly to the present findings that diabetes-induced upregulation of fibronectin and α3Col4 is dependent on Smad3 function.

Enhanced Expression of Osteopontin in Human Diabetic Artery and Analysis of Its Functional Role in Accelerated Atherogenesis

We have previously reported that high glucose stimulates osteopontin (OPN) expression through protein kinase C-dependent pathways as well as hexosamine pathways in cultured rat aortic smooth muscle cells. The finding prompted us to study in vivo expression of OPN in diabetes mellitus. In the present study, we found by immunohistochemistry that medial layers of the carotid arteries of streptozotocin-induced diabetic rats and the forearm arteries of diabetic patients stained positively for OPN antibodies, whereas the staining from arteries of control rats and nondiabetic patients was negative. We also found that OPN stimulated the migration and enhanced platelet-derived growth factor (PDGF)-mediated DNA synthesis of cultured rat aortic smooth muscle cells. OPN and PDGF synergistically activated focal adhesion kinase as well as extracellular signal-regulated kinase; this finding seems to explain the OPN-induced enhancement of PDGF-mediated DNA synthesis. Taken together, our present results raise a possibility that OPN plays a role in the development of diabetic vascular complications.

High Glucose-Induced Upregulation of Osteopontin Is Mediated via Rho/Rho Kinase Pathway in Cultured Rat Aortic Smooth Muscle Cells

Objective—Osteopontin is upregulated in the diabetic vascular wall and in vascular smooth muscle cells cultured under high glucose concentration. In the present study, we analyzed the mechanism of high glucose-induced upregulation of osteopontin in cultured rat aortic smooth muscle cells. Methods and Results—We found that an inhibitor of Rho-associated protein kinase, Y-27632, suppressed osteopontin mRNA expression under high glucose concentration. Transfection of cells with a constitutive active Rho mutant, pSR&agr;-myc-RhoDA, enhanced osteopontin mRNA expression. Furthermore, incubation of cells under high glucose concentration activated Rho, indicating that Rho/Rho kinase pathway mediates high-glucose–stimulated osteopontin expression. Treatment of cells with an inhibitor of protein kinase C, GF109203X, and azaserine, an inhibitor of the hexosamine pathway, suppressed high glucose-induced Rho activation. Glucosamine treatment was shown to activate Rho. Treatment of cells with an inhibitor of MEK1, PD98059, suppressed osteopontin mRNA expression under high glucose concentration. Incubation of cells under high glucose concentration activated ERK. Finally, transfection of cells with pSR&agr;-myc-RhoDA also activated ERK. Conclusions—In conclusion, our present findings support a notion that Rho/Rho kinase pathway functions downstream of protein kinase C and the hexosamine pathways and upstream of ERK in mediating high-glucose–induced upregulation of osteopontin expression.

Targeted Disruption of TGF-β–Smad3 Signaling Leads to Enhanced Neointimal Hyperplasia With Diminished Matrix Deposition in Response to Vascular Injury

The role of transforming growth factor (TGF)-&bgr; and its signal in atherogenesis is not fully understood. Here, we examined mice lacking Smad3, a major downstream mediator of TGF-&bgr;, to clarify the precise role of Smad3-dependent signaling in vascular response to injury. Femoral arteries were injured in wild-type and Smad3-null (null) male mice on C57Bl/6 background. Histopathological evaluation of the arteries 1 to 3 weeks after the injury revealed significant enhancement of neointimal hyperplasia in null compared with wild-type mice. Transplantation of null bone marrow to wild-type mice did not enhance neointimal thickening, suggesting that vascular cells in situ play a major role in the response. Null intima contained more proliferating smooth muscle cells (SMC) with less amount of collagen compared with wild-type intima. TGF-&bgr; caused significant inhibition of cellular proliferation in wild-type aortic SMC, whereas the growth of null SMC was only weakly inhibited by TGF-&bgr; in vitro, indicating a crucial role of Smad3 in the growth inhibitory function. On the other hand, Smad3-deficiency did not attenuate chemotaxis of SMC toward TGF-&bgr;. TGF-&bgr; increased transcript level of &agr;2 type I collagen and tissue inhibitor of metalloproteinases-1, and suppressed expression and activity of matrix metalloproteinases in wild-type SMC. However, these effects of TGF-&bgr; were diminished in null SMC. Our findings altogether show that the loss of Smad3 pathway causes enhanced neointimal hyperplasia on injury through modulation of growth and matrix regulation in vascular SMC. These results indicate a vasculoprotective role of endogenous Smad3 in response to injury.

Suppression of neointimal thickening by a newly developed HMG-CoA reductase inhibitor, BAYw6228, and its inhibitory effect on vascular smooth muscle cell growth

The aim of this study was to determine whether BAYw6228 (BAYw), a newly developed 3‐hydroxy‐3‐methylglutaryl‐CoA (HMG‐CoA) reductase inhibitor, could suppress an atherogenic process such as intimal thickening by a mechanism other than lowering the level of serum cholesterol. First, we evaluated the in vitro effect of BAYw on the proliferation of vascular smooth muscle cells (SMC) from various species: Sprague‐Dawley (SD) rats, New Zealand (NZ) white rabbits, intimal cells from Watanabe hereditary hyperlipidemic (WHHL) rabbit and SMC from the new‐born human aorta. The increasing rate of total protein content of these cells was inhibited by the addition of BAYw in a dose‐dependent fashion. In the presence of 2% foetal calf serum (FCS), the value of IC50 was 1.0 μm in SD rats. 2.1 μm in NZ white rabbits, and 0.3 μm in WHHL rabbits. With human SMC, the value was 0.02 μm in the presence of 10% FCS and 0.2 μm with a mixture of growth factors. Based on these above in vitro findings, we next examined the in vivo effect of the agent to determine whether it could suppress rabbit intimal thickening induced by balloon catheterization. A balloon catheter was inserted from a peripheral branch of the left external carotid artery to the aorta to denude the endothelium of the left common carotid artery in Japanese white rabbits. After 12 days they were divided into control and BAYw groups. The former were subcutaneously injected with saline and the latter with BAYw 1 mg kg−1 day−1. Two days after the beginning of treatment, a second balloon injury was performed to the previously injured left common carotid artery in both groups. After another two weeks, the left common carotid artery was removed and variously stained. Although the total serum cholesterol in the BAYw group was significantly lower than in the control (P<0.05), the difference was not enough to affect intimal thickening. In addition, the BAYw group had a smaller intima/media ratio than the control group, decreasing to 45% of control (P<0.05). By anti‐α smooth muscle actin antibody staining, these intimal thickening areas were entirely occupied by SMCs, and their amount was attenuated by BAYw. By anti‐rabbit macrophage antibody (RAM 11) staining, the number of positive cells in the intimal thickening was markedly decreased in the BAYw group compared to control (P<0.01). These results indicate that BAYw has an inhibitory effect on intimal thickening by attenuating intimal SMC proliferation and infiltration of macrophages, suggesting that BAYw could be effective in the prevention of the progression of atherosclerotic plaque‐like restenosis after angioplasty.

Diagnostic criteria for Werner syndrome based on Japanese nationwide epidemiological survey

Aim:  Werner syndrome (WS) is an autosomal recessive disorder of progeroid symptoms and signs. It is caused by mutations in the WRN gene, which encodes a RecQ DNA helicase. The aim of this study was to revise the diagnostic criteria for Japanese Werner syndrome.

Hyperglycemia-induced alteration of vascular smooth muscle phenotype

We have previously reported that high glucose stimulates osteopontin (OPN) expression through protein kinase C-dependent pathway, as well as the hexosamine pathway, in cultured rat aortic smooth muscle cells (SMC). The finding prompted us to study in vivo expression of OPN in diabetes mellitus. In the present study, we found by immunohistochemistry that medial layers of the carotid arteries of streptozotocin (STZ)-induced diabetic rats, as well as the forearm arteries of diabetic patients, stained positive with OPN antibodies, whereas the staining of control rats, as well as nondiabetic patients, was negative. We also found that OPN stimulated migration and enhanced platelet-derived growth factor (PDGF)-mediated DNA synthesis of cultured rat aortic SMC. OPN and PDGF synergistically activated focal adhesion kinase (FAK), as well as extracellular signal-regulated kinase (ERK), which seems to be a reason for OPN-induced enhancement of PDGF-mediated DNA synthesis. Taken together, our present results raise a possibility that OPN plays a role in the development of diabetic vascular complications.

Influence of C-peptide on early glomerular changes in diabetic mice

C‐peptide has been shown to ameliorate diabetes‐induced functional and structural renal changes in animal models as well as in patients with type 1 diabetes. This study aims to examine the molecular effects of C‐peptide on early glomerular changes in a mouse model of type 1 diabetes.

NK-104, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, reduces osteopontin expression by rat aortic smooth muscle cells.

It has been suggested that osteopontin promotes the development of atherosclerosis, especially under diabetic conditions. In the present study, we found that NK‐104, a new potent synthetic inhibitor of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase, reduced osteopontin expression both at protein and mRNA levels in cultured rat aortic smooth muscle cells. The inhibitory effect of NK‐104 was almost completely reversed by mevalonate, suggesting that mevalonate or its metabolites play important roles in the regulation of osteopontin expression. Furthermore, oral administration of NK‐104 (3 mg kg−1 day−1 for 7 days) effectively suppressed abnormally upregulated expression of osteopontin mRNA in the aorta and kidney of streptozotocin‐induced diabetic rats. These data support a notion that NK‐104 is a suitable drug for the treatment of diabetic patients with hypercholesterolaemia.