Fasudil hydrochloride induces osteoblastic differentiation of stromal cell lines, C3H10T1/2 and ST2, via bone morphogenetic protein-2 expression

Kanazawa, Ippei; Yamaguchi, Toru; Yano, Shozo; Yamauchi, Mika; Sugimoto, Toshitsugu

doi:10.1507/endocrj.k09e-328

Cited by 12 publications

(7 citation statements)

References 34 publications

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“…This increase in ALP upon ROCK inhibition is, in fact, consistent with recent findings describing the mineralization of mesenchymal cells and vascular SMCs after treatment with a ROCK inhibitor (5,23). Specifically, the application of fasudil or Y27632 to stromal stem cell lines or SMCs, respectively, resulted in a significant elevation of ALP (5,23) and mineralization through ossific mechanisms (23). In our work, the concomitant decrease in apoptotic cells with an increase in osteoblastic activity may indicate that many of the nodules formed on cultures treated with H1152 were via osteogenic more so than dystrophic mechanisms.…”

Section: Discussionsupporting

confidence: 92%

Role of the Rho pathway in regulating valvular interstitial cell phenotype and nodule formation

Gu¹,

Masters

2011

American Journal of Physiology-Heart and Circulatory Physiology

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The differentiation of valvular interstitial cells (VICs) to a myofibroblastic or osteoblast-like phenotype is commonly found in calcific valvular stenosis, although the molecular-level mechanisms of this process remain poorly understood. Due to the role of the Rho pathway in various vascular diseases and in the expression of a myofibroblast phenotype, the present study was inspired by the hypothesis that Rho activation is involved in regulating cellular processes related to valve calcification. It was found that increased RhoA and Rho kinase (ROCK) activity was associated with increased nodule formation in VIC cultures in vitro, and intentional induction of RhoA activity led to a further increase in nodules and expression of α-smooth muscle actin. VICs treated with ROCK inhibitors were also examined for nodule formation, proliferation, apoptosis, and expression of myofibroblastic or osteoblastic markers. ROCK inhibition dramatically reduced myofibroblast-regulated nodule formation in VIC cultures, as evidenced by a decrease in nodule number, total nodule area, α-smooth muscle actin-positive stress fibers, apoptosis, and gene expression of myofibroblast-related phenotypic markers. Meanwhile, ROCK inhibition was less effective at reducing nodule formation associated with osteogenic activity. In fact, ROCK inhibition increased the expression of alkaline phosphatase and effected only a modest decrease in nodule number when applied to VIC cultures with higher osteogenic activity. Thus, the Rho pathway possesses a complex role in regulating the VIC phenotype and nodule formation, and it is hoped that further elucidation of these molecular-level events will lead to an improved understanding of valvular disease and identification of potential treatments.

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Section: Discussionsupporting

confidence: 92%

Role of the Rho pathway in regulating valvular interstitial cell phenotype and nodule formation

Gu¹,

Masters

2011

American Journal of Physiology-Heart and Circulatory Physiology

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“…Interestingly, Runx2 gene expression levels did not change under Rac or ROCK inhibition (data not shown), suggesting that upregulation of the gene is not required and it is nuclear accumulation of the protein that is altered. ROCK inhibition has been previously demonstrated to increase osteocalcin synthesis and biomineralization of mouse calvarial osteoblasts [45], [46] and the stromal cell lines C3H10T1/2 and ST2 [47] cultured on tissue culture polystyrene. Furthermore, Yoskihawa et al, 2009, observed that ROCK inhibition induced ectopic bone formation in response to rhBMP-2 [48].…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Rac and ROCK Signalling Influence Osteoblast Adhesion, Differentiation and Mineralization on Titanium Topographies

et al. 2013

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Reducing the time required for initial integration of bone-contacting implants with host tissues would be of great clinical significance. Changes in osteoblast adhesion formation and reorganization of the F-actin cytoskeleton in response to altered topography are known to be upstream of osteoblast differentiation, and these processes are regulated by the Rho GTPases. Rac and RhoA (through Rho Kinase (ROCK)). Using pharmacological inhibitors, we tested how inhibition of Rac and ROCK influenced osteoblast adhesion, differentiation and mineralization on PT (Pre-treated) and SLA (sandblasted large grit, acid etched) topographies. Inhibition of ROCK, but not Rac, significantly reduced adhesion number and size on PT, with adhesion size consistent with focal complexes. After 1 day, ROCK, but not Rac inhibition increased osteocalcin mRNA levels on SLA and PT, with levels further increasing at 7 days post seeding. ROCK inhibition also significantly increased bone sialoprotein expression at 7 days, but not BMP-2 levels. Rac inhibition significantly reduced BMP-2 mRNA levels. ROCK inhibition increased nuclear translocation of Runx2 independent of surface roughness. Mineralization of osteoblast cultures was greater on SLA than on PT, but was increased by ROCK inhibition and attenuated by Rac inhibition on both topographies. In conclusion, inhibition of ROCK signalling significantly increases osteoblast differentiation and biomineralization in a topographic dependent manner, and its pharmacological inhibition could represent a new therapeutic to speed bone formation around implanted metals and in regenerative medicine applications.

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“…SYBR Green chemistry was used to perform quantitative determination of the mRNA of peroxisome proliferator-activated receptor γ (PPARγ) and a housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), following an optimized protocol (12)(13)(14). Briefly, total RNA was isolated with TRIzol reagent (Invitrogen, San Diego, CA, USA) and further cleaned by two successive phenol/ chloroform extractions.…”

Section: Real-time Pcr Quantification Of Gene Expressionmentioning

confidence: 99%

Effects of Uremic Toxin p‐Cresol on Proliferation, Apoptosis, Differentiation, and Glucose Uptake in 3T3‐L1 Cells

et al. 2014

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Malnutrition is a common feature seen in chronic dialysis patients, and the survival rate of obese patients receiving such treatment is higher than that of lean patients. Irrespective of obesity or diabetes, dialysis patients commonly have insulin resistance, and the leading cause of death is cardiovascular (CV) disease. It has been reported that the concentration of p-cresol, a uremic toxin, is highly associated with CV events. As uremic toxin levels are high in dialysis patients, they may be involved in the pathogenesis of insulin resistance and CV disease in this population. However, little is known so far. Thus, we focused on this uremic toxin to examine its effects on adipocytes and their precursors. 3T3-L1 cells, a mouse preadipocyte cell line, were cultured until 90% confluency. The cells were then differentiated with 500 μM 3-isobutyl-methylxanthine, 250 nM dexamethasone, and 10 μg/mL insulin. Cell proliferation was evaluated by cell counting and bromodeoxyuridine (Brd-U) incorporation assay. Glucose uptake was estimated using radiolabeled 2-deoxyglucose. The range of concentrations of p-cresol used in the experiments was from 2 to 200 μM. The investigation of cell proliferation by cell counting revealed that, compared with control, 3T3-L1 cells treated with 100 and 200 μM p-cresol were significantly decreased in number at day 3 and day 7 of culture. The Brd-U incorporation assay also demonstrated similar inhibitory effects on cell proliferation, suggesting that p-cresol affected the normal cell cycle. Oil Red O staining at day 7 showed that the number of mature adipocytes was decreased by treatment with 200 μM p-cresol. Consistent with that finding, the number of apoptotic cells at day 7 was increased by treatment with 100 and 200 μM p-cresol. Peroxisome proliferator-activated receptor γ (PPARγ) mRNA expression increased time-dependently during the differentiation process of 3T3-L1 cells. p-Cresol dose-dependently decreased differentiation-induced mRNA expression of PPARγ. Uptake of 3H-labeled 2-deoxyglucose was markedly decreased by 200 μM p-cresol in the presence or in the absence of insulin, mainly because of the decreased number of mature adipocytes. High concentrations of p-cresol disturbed the cell cycle, induced apoptosis, inhibited the differentiation of preadipocytes into mature adipocytes, and decreased glucose uptake at baseline and after insulin stimulation. These findings indicate that accumulated p-cresol may induce reduction in adipose tissue, insulin resistance, and malnutrition, eventually leading to poor outcomes in chronic dialysis patients.

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Fasudil hydrochloride induces osteoblastic differentiation of stromal cell lines, C3H10T1/2 and ST2, via bone morphogenetic protein-2 expression

Cited by 12 publications

References 34 publications

Role of the Rho pathway in regulating valvular interstitial cell phenotype and nodule formation

Role of the Rho pathway in regulating valvular interstitial cell phenotype and nodule formation

Inhibition of Rac and ROCK Signalling Influence Osteoblast Adhesion, Differentiation and Mineralization on Titanium Topographies

Effects of Uremic Toxin p‐Cresol on Proliferation, Apoptosis, Differentiation, and Glucose Uptake in 3T3‐L1 Cells

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