Toshifumi Azuma

Side population of pancreatic cancer cells predominates in TGF-β-mediated epithelial to mesenchymal transition and invasion

We report here side population (SP) cells, a cancer stem cell enriched fraction from pancreatic cancer cell line, have enormous superior potential of the epithelial to mesenchymal transition (EMT), invasion, and metastasis. In an isolated SP cell culture, the cells rapidly expressed and up‐regulated E‐cadherin, an epithelial phenotypic marker, and the cells formed tightly contacted cell cluster, which is a representative epithelial phenotypic appearance. When the SP cells were incubated in the presence of TGF‐β, SP cells changed their shape into mesenchymal‐like appearance including spindle shaped assembly. This alteration was associated with significant reduction of E‐cadherin expression level. TGF‐β induced EMT‐associated gene alteration such as reduction of E‐cadherin mRNA and induction of Snail mRNA and matrixmetalloproteinase (MMP)‐2 mRNA. Finally, SP cells exerted notable matrigel invasion activity in response to TGF‐β treatment, whereas MP cells did not respond to TGF‐β‐mediated invasion. In conclusion, these results suggest that SP cells from pancreatic cancer cell line possess superior potentials of phenotypic switch, i.e., EMT/MET, micro‐invasion, and in vivo metastasis, as compared to MP cells. Because micro‐invasion and metastasis are key mechanisms of cancer malignant potential, SP cells would be the attractive target for preventing cancer progression.

Inhibition of Insulin-like Growth Factor-1 (IGF-1) Expression by Prolonged Transforming Growth Factor-β1 (TGF-β1) Administration Suppresses Osteoblast Differentiation

Background: TGF-β1 positively and negatively regulates osteoblast differentiation. Results: Repeated TGF-β1 negatively regulates osteoblast differentiation caused by inhibiting IGF-1 expression and Akt phosphorylation. Conclusion: Prolonged TGF-β1 treatment inhibits osteoblast differentiation of mesenchymal stem cells via the suppression of the IGF-1 signaling pathway. Significance: IGF-1 administration may recover the suppression of osteogenesis and promotion of bone resorption due to chronic inflammation by TGF-β1. TGF-β1 can regulate osteoblast differentiation not only positively but also negatively. However, the mechanisms of negative regulation are not well understood. We previously established the reproducible model for studying the suppression of osteoblast differentiation by repeated or high dose treatment with TGF-β1, although single low dose TGF-β1 strongly induced osteoblast differentiation. The mRNA expression and protein level of insulin-like growth factor-1 (IGF-1) were remarkably decreased by repeated TGF-β1 administration in human periodontal ligament cells, human mesenchymal stem cells, and murine preosteoblast MC3T3-E1 cells. Repeated TGF-β1 administration subsequently decreased alkaline phosphatase (ALP) activity and mRNA expression of osteoblast differentiation marker genes, such as RUNX2, ALP, and bone sialoprotein (BSP). Additionally, repeated administration significantly reduced the downstream signaling pathway of IGF-1, such as Akt phosphorylation in these cells. Surprisingly, exogenous and overexpressed IGF-1 recovered ALP activity and mRNA expression of osteoblast differentiation marker genes even with repeated TGF-β1 administration. These facts indicate that the key mechanism of inhibition of osteoblast differentiation induced by repeated TGF-β1 treatment is simply due to the down-regulation of IGF-1 expression. Inhibition of IGF-1 signaling using small interfering RNA (siRNA) against insulin receptor substrate-1 (IRS-1) suppressed mRNA expression of RUNX2, ALP, BSP, and IGF-1 even with single TGF-β1 administration. This study showed that persistence of TGF-β1 inhibited osteoblast differentiation via suppression of IGF-1 expression and subsequent down-regulation of the PI3K/Akt pathway. We think this fact could open the way to use IGF-1 as a treatment tool for bone regeneration in prolonged inflammatory disease.

Akt Activation is Required for TGF-β1-Induced Osteoblast Differentiation of MC3T3-E1 Pre-Osteoblasts

Background We have previously reported that repeated treatment of human periodontal ligament cells and murine pre-osteoblast MC3T3-E1 cells with transforming growth factor-beta 1 (TGF-β1) inhibited their osteoblastic differentiation because of decreased insulin-like growth factor-1 (IGF-1) secretion. We also found that IGF-1/PI3K signaling plays an important role in osteoblast differentiation induced by TGF-β1 treatment; however, the downstream signaling controlling this remains unknown. The aim of this current study is to investigate whether Akt activation is required for osteoblast differentiation. Methodology/Principal Findings MC3T3-E1 cells were cultured in osteoblast differentiation medium (OBM) with or without 0.1 ng/mL TGF-β1. OBM containing TGF-β1 was changed every 12 h to provide repeated TGF-β1 administration. MC3T3-E1 cells were infected with retroviral vectors expressing constitutively active (CA) or dominant-negative (DN)-Akt. Alkaline phosphatase (ALP) activity and osteoblastic marker mRNA levels were substantially decreased by repeated TGF-β1 treatment compared with a single TGF-β1 treatment. However, expression of CA-Akt restored ALP activity following TGF-β1 treatment. Surprisingly, ALP activity increased following multiple TGF-β1 treatments as the number of administrations of TGF-β1 increased. Activation of Akt significantly enhanced expression of osteocalcin, but TGF-β1 treatment inhibited this. Mineralization of MC3T3-E1 cells was markedly enhanced by CA-Akt expression under all medium conditions. Exogenous IGF-1 restored the down-regulation of osteoblast-related gene expression by repeated TGF-β1 administration. However, in cells expressing DN-Akt, these levels remained inhibited regardless of IGF-1 treatment. These findings indicate that Akt activation is required for the early phase of osteoblast differentiation of MC3T3-E1 cells induced by TGF-β1. However, Akt activation is insufficient to reverse the inhibitory effects of TGF-β1 in the late stages of osteoblast differentiation. Conclusions TGF-β1 could be an inducer or an inhibitor of osteoblastic differentiation of MC3T3-E1 cells depending on the state of Akt phosphorylation. Our results indicate that Akt is the molecular switch for TGF-β1-induced osteoblastic differentiation of MC3T3-E1 cells.

A novel strategy for enrichment and isolation of osteoprogenitor cells from induced pluripotent stem cells based on surface marker combination.

In this study, we developed a new method to stimulate osteogenic differentiation in tissue-nonspecific alkaline phosphatase (TNAP)-positive cells liberated from human induced pluripotent stem cells (hiPSCs)-derived embryoid bodies (EBs) with 14 days long TGF-β/IGF-1/FGF-2 treatment. TNAP is a marker protein of osteolineage cells. We analyzed and isolated TNAP-positive and E-cadherin-negative nonepithelial cells by fluorescence-activated cell sorting. Treating the cells with a combination of transforming growth factor (TGF)-β, insulin-like growth factor (IGF)-1, and fibroblast growth factor (FGF)-2 for 14 days greatly enhanced TNAP expression and maximized expression frequency up to 77.3%. The isolated cells expressed high levels of osterix, which is an exclusive osteogenic marker. Culturing these TNAP-positive cells in osteoblast differentiation medium (OBM) led to the expression of runt-related transcription factor 2, type I collagen, bone sialoprotein, and osteocalcin (OCN). These cells responded to treatment with activated vitamin D3 by upregulating OCN. Furthermore, in OBM they were capable of generating many mineralized nodules with strong expression of receptor activator of NF-kappaB ligand and sclerostin (SOST). Real-time RT-PCR showed a significant increase in the expression of osteocyte marker genes, including SOST, neuropeptide Y, and reelin. Scanning electron microscopy showed dendritic morphology. Examination of semi-thin toluidine blue-stained sections showed many interconnected dendrites. Thus, TNAP-positive cells cultured in OBM may eventually become terminally differentiated osteocyte-like cells. In conclusion, treating hiPSCs-derived cells with a combination of TGF-β, IGF-1, and FGF-2 generated TNAP-positive cells at high frequency. These TNAP-positive cells had a high osteogenic potential and could terminally differentiate into osteocyte-like cells. The method described here may reveal new pathways of osteogenesis and provide a novel tool for regenerative medicine and drug development.

Transforming growth factor β and Ras/MEK/ERK signaling regulate the expression level of a novel tumor suppressor lefty

Objectives The objectives of the present study were (i) to identify a novel tumor suppressor gene whose expression level was regulated by transforming growth factor (TGF-&bgr;) and (ii) to evaluate the effect of Ras/MEK/ERK signaling on TGF-&bgr;–dependent Lefty up-regulation. Methods Human pancreatic cancer cell lines were used. The effect of Ras/MEK/ERK pathway on TGF-&bgr;–mediated Lefty up-regulation was tested by adding K-ras small interfering RNA, MEK inhibitor U0126, or extracellular signal–regulated kinase (ERK) inhibitor LY294002. Results Transforming growth factor &bgr; upregulated Lefty messenger RNA levels within 6 of the 7 cell lines. Lefty exerts an antagonistic effect against the tumor-promoting molecule, Nodal, as recombinant Lefty suppressed Nodal-mediated proliferation. Interestingly, inhibition of the Ras/MEK/ERK pathway dramatically enhanced TGF-mediated Lefty up-regulation, suggesting that Ras/MEK/ERK signaling suppresses TGF-&bgr;–Lefty pathway. Conclusions Our data suggest that Lefty is a novel TGF-&bgr; target molecule that mediates growth inhibition of pancreatic cancer cells. In addition, activation of the Ras/MEK/ERK pathway serves as a mechanism by which pancreatic cancer escapes from growth inhibition by the TGF-&bgr;–Lefty axis. The results imply a novel therapeutic strategy for pancreatic cancer, that is, combination treatment with Ras/MEK/ERK inhibitors and TGF-&bgr;.

Promoting effect of 1,25(OH)2 vitamin D3 in osteogenic differentiation from induced pluripotent stem cells to osteocyte-like cells.

We recently reported a new method to purify the induced pluripotent stem (iPS)-derived osteoprogenitors (iPSop). In this paper, we optimized the procedure and characterized cells at each process step. We observed that 10 days of treatment with FGF-2, IGF-1 and TGF-β (FIT) resulted in early-phase osteoblasts and 14 days of treatment resulted in late-phase osteoblasts. We found that treatment with 1,25(OH)2 vitamin D3 increased expression of osteocalcin and decreased expression of tissue-non-specific alkaline phosphatase and runt-related transcription factor 2 (RUNX2) in iPSop-day14 cells (cells treated with FIT for 14 days). Therefore, iPSop-day14 cells were promoted to mature osteoblasts by 1,25(OH)2 vitamin D3 treatment. In addition, we found that 1,25(OH)2 vitamin D3 treatment for 14 days enhanced not only mineralization but also expression of osteocyte markers, including dentin matrix protein-1 and fibroblast growth factor-23, in iPSop cells. Therefore, 1,25(OH)2 vitamin D3 is a potent promoter of osteoblast–osteocyte transition. The results of this study suggest that it is possible to evaluate both early- and late-phase osteoblasts and to apply cells to drug screening for anabolic drugs that stimulate bone formation.

Suppression of Lefty expression in induced pluripotent cancer cells

Cancer and stem cells share the ability to silence tumor suppressors. We focused on Lefty, which encodes one of the most abundant tumor suppressors in embryonic stem (ES) cells and is not expressed in somatic cancer cells. We found that transforming growth factor β (TGF‐β) induced demethylation of the Lefty B cytosine‐phosphate‐guanine (CpG) island and increased Lefty expression (10–200 times) in human pancreatic cancer cells and human liver cancer cells (PLC/PRF/5 and HLF). Expression of Cripto, another important factor in Nodal‐Lefty signaling, was not increased after adding TGF‐β. We generated reprogrammed cancer cells that revealed high expression of immature marker proteins, high proliferation, and the potential to express morphological patterns of ectoderm, mesoderm, and endoderm, suggesting that these cells may have cancer stem cell‐like phenotypes. We investigated Lefty and found that reprogrammed human liver cancer cells (induced pluripotent cancer cells) displayed a much lower ability to express Lefty, although less Lefty B CpG methylation was also observed. We also found that a MEK inhibitor dramatically enhanced Lefty expression in human pancreatic cancers with mutated ras, whereas Lefty B CpG methylation was not decreased. These observations indicate that despite the demethylation of DNA strands in promoter regions of pluripotency‐associated genes, including Lefty gene, Lefty expression was not induced well in reprogrammed cells. Of note was the fact that Lefty is abundantly expressed in human ES cells but not in induced pluripotent stem (iPS) cells. We thus think that reprogrammed cancer cells share the mechanism for expression of Lefty with iPS cells. This shared mechanism may contribute to the cancerous transformation of iPS cells.—Saito, A., Ochiai, H., Okada, S., Miyata, N., Azuma, T. Suppression of Lefty expression in induced pluripotent cancer cells. FASEB J. 27, 2165–2174 (2013). www.fasebj.org

The malQ gene is essential for starch metabolism in Streptococcus mutans

Background The malQ and glgP genes, respectively, annotated as putative 4-α-glucanotransferase and putative glycogen phosphorylase are located with a 29 nucleotide overlap on the Streptococcus mutans genome. We found that the glgP gene of this organism was induced with maltose, and the gene likely constituted an operon with the upstream gene malQ. This putative operon was negatively regulated with the malR gene located upstream from the malQ gene and a MalR-binding consensus sequence was found upstream of the malQ gene. S. mutans is not able to catabolize starch. However, this organism utilizes maltose degraded from starch in the presence of saliva amylase. Therefore, we hypothesized that the MalQ/GlgP system may participate in the metabolism of starch-degradation products. Methods A DNA fragment amplified from the malQ or glgP gene overexpressed His-tagged proteins with the plasmid pBAD/HisA. S. mutans malQ and/or glgP mutants were also constructed. Purified proteins were assayed for glucose-releasing and phosphorylase activities with appropriate buffers containing maltose, maltotriose, maltodextrin, or amylodextrin as a substrate, and were photometrically assayed with a glucose-6-phosphate dehydrogenase–NADP system. Results Purified MalQ protein released glucose from maltose and maltotriose but did not from either maltodextrin or amylodextrin. The purified GlgP protein did not exhibit a phosphorylase reaction with maltose or maltotriose but generated glucose-1-phosphate from maltodextrin and amylodextrin. However, the GlgP protein released glucose-1-phosphate from maltose and maltotriose in the presence of the MalQ protein. In addition, the MalQ enzyme activity with maltose released not only glucose but also produced maltooligosaccharides as substrates for the GlgP protein. Conclusion These results suggest that the malQ gene encodes 4-α-glucanotransferase but not α-1,4-glucosidase activity. The malQ mutant could not grow in the presence of maltose as a carbon source, which suggests that the malQ gene is essential for the utilization of starch-degradation products.

Transplantation of human-induced pluripotent stem cells carried by self-assembling peptide nanofiber hydrogel improves bone regeneration in rat calvarial bone defects

Objectives/Aims:The requisite conditions for successful bone tissue engineering are efficient stem cell differentiation into osteogenic cells and a suitable scaffold. In this study, we investigated in vivo bone regeneration from transplanted induced pluripotent stem cells (iPSCs).Materials and Methods:Two critical-sized calvarial bone defects were created in 36 rats. The surgical sites were randomly assigned to one of three treatments to test the healing effectiveness of the scaffold alone, scaffold with iPSCs or a salt solution as a control. The effectiveness of the treatments was evaluated after 2 or 4 weeks using radiographic and histological analyses of bone regeneration in the six groups.Results:Micro-computed tomography (CT) analysis of the bone defects found minimal bone regeneration with the salt solution and nanofiber scaffold and increased bone regeneration in defects repaired with iPSCs delivered in the nanofiber scaffold.Conclusion:Transplanted iPSCs encapsulated in a nanofiber scaffold can regenerate bone in critical-sized defects.

Glucose-PTS Involvement in Maltose Metabolism by Streptococcus mutans.

Streptococcus mutans grows with starch-derived maltose in the presence of saliva. Maltose transported into the cells is mediated by the MalQ protein (4-alpha-glucanotransferase) to produce glucose and maltooligosaccharides. Glucose can be phosphorylated to glucose 6-phosphate, which can enter the glycolysis pathway. The MalQ enzyme is essential in the catabolism of maltose when it is the sole carbon source, suggesting the presence of a downstream glucokinase of the MalQ enzyme reaction. However, a glucokinase gene-inactivated mutant (glk mutant) grew with maltose as the sole carbon source, with no residual glucokinase activity. This left a phosphoenolpyruvate-dependent phosphotransferase system (PTS) as the only candidate pathway for the phosphorylation of glucose in its transport as a substrate. Our hypothesis was that intracellular glucose derived from maltose mediated by the MalQ protein was released into the extracellular environment, and that such glucose was transported back into the cells by a PTS. The mannose PTS encoded by the manL, manM, and manN genes transports glucose into cells as a high affinity system with concomitant phosphorylation. The purpose of this study was to investigate extracellular glucose by using an enzyme-linked photometrical method, monitoring absorbance changes at 340 nm in supernatant of S. mutans cells. A significant amount of glucose was detected in the extracellular fluid of a glk, manLM double mutant. These results suggest that the glk and manLMN genes participate in maltose catabolism in this organism. The significance of multiple metabolic pathways for important energy sources, including maltose, in the oral environment is discussed.