mRNA Expression Of Differentiation Markers Research Articles

Terminal differentiation of keratinocytes was damaged in type 2 diabetic mice.

Although skin manifestations are common in diabetic patients, its characteristics are poorly identified. This study explored the differentiation process of keratinocytes in type 2 diabetes mellitus (T2DM) in vivo. Back skin of T2DM model KKAy/TaJcl mice (KKAy) and C57BL/6JJcl mice (control) aged 8 and 12weeks was used. The mRNA expression of differentiation markers of keratinocytes was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of each marker in situ was examined immunohistochemically. KKAy mice showed hyperglycemia versus control mice. The histological findings showed increased thickness and structural impairment of epidermal tissue in KKAy mice. The qRT-PCR revealed that the expression of integrin beta 1 and keratin 14 in KKAy and control mice was identical. However, the expression of involucrin at 8 weeks, keratin 10 at 12 weeks, and filaggrin and loricrin at 8 and 12 weeks was decreased in KKAy mice. Immunohistochemical findings showed that filaggrin was markedly decreased in KKAy mice, though Ki-67 remained unchanged. The terminal differentiation process was impaired in the diabetic skin, while keratinocyte proliferation was preserved. Damaged terminal differentiation of keratinocytes may contribute to impairment of the skin barrier function in diabetic dermatoses.

Vaccinium virgatum Aiton Leaves Extract Suppressed Lipid Accumulation and Uric Acid Production in 3T3-L1 Adipocytes.

Blueberry (Vaccinium virgatum Aiton; Kinisato 35 Gou) leaves have recently attracted increasing attention as a useful material for the prevention of lifestyle diseases. Here, we examined the effects of the hot water extract of blueberry leaves (BLEx) on lipogenesis and uric acid production in 3T3-L1 adipocytes. The results showed that BLEx suppressed lipid accumulation and the mRNA expression of differentiation markers in 3T3-L1 adipocytes. A fractionation study showed that the highly polymerized proanthocyanidin-rich fraction was responsible for this effect. Upon maturation to adipocytes, 3T3-L1 cells produced uric acid and tumor necrosis factor-α, and hypoxia stimulated the production of uric acid and xanthine oxidoreductase activity. BLEx suppressed the production of uric acid under these conditions. Although BLEx inhibited the enzymatic activity of xanthine oxidase, this activity was observed in several fractions containing catechin, epicatechin, chlorogenic acid, rutin, and low molecular weight proanthocyanidins. Taken together, these results indicate that BLEx contains various compounds with the ability to suppress lipid accumulation and uric acid production in adipocytes.

AICAR Stimulates the Pluripotency Transcriptional Complex in Embryonic Stem Cells Mediated by PI3K, GSK3β, and β-Catenin.

Pluripotent stem cells maintain the property of self-renewal and differentiate into all cell types under clear environments. Though the gene regulatory mechanism for pluripotency has been investigated in recent years, it is still not completely understood. Here, we show several signaling pathways involved in the maintenance of pluripotency. To investigate whether AMPK is involved in maintaining the pluripotency in mouse embryonic stem cells (mESCs) and elucidating the possible molecular mechanisms, implicated D3 and R1/E mESC lines were used in this study. Cells were cultured in the absence or presence of LIF and treated with 1 mM and 0.5 mM 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), 2 mM metformin, compound C, and the PI3K inhibitor LY294002 for 24, 72, and 120 h. The levels of Nanog, Oct3/4, and REX1 and Brachyury, Notch2, and Gata4 mRNAs and Nanog or OCT3/4 protein levels were analyzed. Alkaline phosphatase and the cellular cycle were determined. The pGSK3β, GSK3β, p-β-catenin, and β-catenin protein levels were also investigated. We found that AMPK activators such as AICAR and metformin increase mRNA expression of pluripotency markers and decrease mRNA expression of differentiation markers in R1/E and D3 ES cells. AICAR increases phosphatase activity and arrests the cellular cycle in the G1 phase in these cells. We describe that AICAR effects were mediated by AMPK activation using a chemical inhibitor or by silencing this gene. AICAR effects were also mediated by PI3K, GSK3β, and β-catenin in R1/E ES cells. According to our findings, we provide a mechanism by which AICAR increases and maintains a pluripotency state through enhanced Nanog expression, involving AMPK/PI3K and p-GSK3β Ser21/9 pathways backing up the AICAR function as a potential target for this drug controlling pluripotency. The highlights of this study are that AICAR (5-aminoimidazole-4-carboxamied-1-b-riboside), an AMP protein kinase (AMPK) activator, blocks the ESC differentiation and AMPK is a key enzyme for pluripotency and shows valuable data to clarify the molecular pluripotency mechanism.

MEIS1 knockdown may promote differentiation of esophageal squamous carcinoma cell line KYSE-30.

Background MEIS1 (Myeloid ecotropic viral integration site 1), as a homeobox (HOX) transcription factor, has a dual function in different types of cancer. Although numerous roles are proposed for MEIS1 in differentiation, stem cell function, gastrointestinal development and tumorigenesis, the involved molecular mechanisms are poor understood. Our aim in this study was to elucidate the functional correlation between MEIS1, as regulator of differentiation process, and the involved genes in cell differentiation in human esophageal squamous carcinoma (ESC) cell line KYSE‐30.MethodsThe KYSE‐30 cells were transduced using recombinant retroviral particles containing specific shRNA sequence against MEIS1 to knockdown MEIS1 gene expression. Following RNA extraction and cDNA synthesis, mRNA expression of MEIS1 and the selected genes including TWIST1, EGF, CDX2, and KRT4 was examined using relative comparative real‐time PCR.ResultsRetroviral transduction caused a significant underexpression of MEIS1 in GFP‐hMEIS1 compared to control GFP cells approximately 5.5‐fold. While knockdown of MEIS1 expression caused a significant decrease in EGF and TWIST1 mRNA expression, nearly ‐8‐ and ‐12‐fold respectively, it caused a significant increase in mRNA expression of differentiation markers including KRT4 and CDX2, approximately 34‐ and 1.14‐fold, correspondingly.Conclusion MEIS1 gene silencing in KYSE‐30 cells increased expression of epithelial markers and decreased expression of epithelial‐mesenchymal transition (EMT) marker TWIST1. It may highlight the role of MEIS1 in differentiation process of KYSE‐30 cells. These results may confirm that MEIS1 silencing promotes differentiation and decreases EMT capability of ESC cell line KYSE‐30.

Single-cell differences in matrix gene expression do not predict matrix deposition.

Mesenchymal stem cells (MSCs) display substantial cell-to-cell heterogeneity, complicating their use in regenerative medicine. However, conventional bulk assays mask this variability. Here we show that both chondrocytes and chondrogenically induced MSCs exhibit substantial mRNA expression heterogeneity. Single-molecule RNA FISH to measure mRNA expression of differentiation markers in single cells reveals that sister cell pairs have high levels of mRNA variability, suggesting that marker expression is not heritable. Surprisingly, this variability does not correlate with cell-to-cell differences in cartilage-like matrix production. Transcriptome-wide analysis suggests that no combination of markers can predict functional potential. De-differentiating chondrocytes also show a disconnect between mRNA expression of the cartilage marker aggrecan and cartilage-like matrix accumulation. Altogether, these quantitative analyses suggest that sorting subpopulations based on these markers would only marginally enrich the progenitor population for ‘superior' MSCs. Our results suggest that instantaneous mRNA abundance of canonical markers is tenuously linked to the chondrogenic phenotype at the single-cell level.

Expression of Caveolin-1 in Periodontal Tissue and Its Role in Osteoblastic and Cementoblastic Differentiation In Vitro.

It has been previously reported that caveolin-1 (Cav-1) knockout mice exhibit increased bone size and stiffness. However, the expression and role of Cav-1 on periodontal tissue is poorly understood. The aim of this study was to investigate the immunohistochemical expression of Cav-1 in the mouse periodontium and explore the role of Cav-1 on osteoblastic and cementoblastic differentiation in human periodontal ligament cells (hPDLCs), cementoblasts, and osteoblasts. To reveal the molecular mechanisms of Cav-1 activity, associated signaling pathways were also examined. Immunolocalization of Cav-1 was studied in mice periodontal tissue. Differentiation was evaluated by ALP activity, alizarin red S staining, and RT-PCR for marker genes. Signal transduction was analyzed using Western blotting and confocal microscopy. Cav-1 expression was observed in hPDLCs, cementoblasts, and osteoblasts of the periodontium both in vivo and in vitro. Inhibition of Cav-1 expression by methyl-β-cyclodextrin (MβCD) and knockdown of Cav-1 by siRNA promoted osteoblastic and cementoblastic differentiation by increasing ALP activity, calcium nodule formation, and mRNA expression of differentiation markers in hPDLCs, cementoblasts, and osteoblasts. Osteogenic medium-induced BMP-2 and BMP-7 expression, and phosphorylation of Smad1/5/8 were enhanced by MβCD and siRNA knockdown of Cav-1, which was reversed by BMP inhibitor noggin. MβCD and Cav-1 siRNA knockdown increased OM-induced AMPK, Akt, GSK3β, and CREB phosphorylation, which were reversed by Ara-A, a specific AMPK inhibitor. Moreover, OM-induced activation of p38, ERK, JNK, and NF-κB was enhanced by Cav-1 inhibition. This study demonstrates, for the first time, that Cav-1 is expressed in developing periodontal tissue and in vitro in periodontal-related cells. Cav-1 inhibition positively regulates osteoblastic differentiation in hPDLCs, cementoblasts, and osteoblasts via BMP, AMPK, MAPK, and NF-κB pathway. Thus, Cav-1 inhibition may be a novel molecular target for therapeutic approaches in periodontitis or osteolytic disease.

Expression of thymosin beta-4 in human periodontal ligament cells and mouse periodontal tissue and its role in osteoblastic/cementoblastic differentiation

A recent report showed that thymosin beta-4 (Tβ4) is expressed during the development of tooth germ, but its effect on osteoblastic/cementoblastic differentiation is a controversial topic. Furthermore, the precise expression and function of Tβ4 in periodontal tissue remains unclear. Therefore, the purpose of this study was to investigate the immunolocalization of Tβ4 in the developing periodontium of mouse, the function of Tβ4 in osteoblastic/cementoblastic differentiation, and the underlying mechanism regulating periodontal regeneration in human periodontal ligament cells (hPDLCs), cementoblasts, and osteoblasts.Tβ4 expression was observed in differentiating hPDLCs, osteoblasts of the periodontium during development, as well as in mature tissue. Higher Tβ4 expression was observed in hPDLCs than in cementoblasts and osteoblasts in the developing periodontium. The expression of Tβ4 mRNA and protein gradually increased during PDL cell differentiation. The downregulation of Tβ4 expression by Tβ4 siRNA transfection inhibited osteoblastic differentiation by decreasing calcium nodule formation, alkaline phosphatase (ALP) activity, and mRNA expression of differentiation markers in hPDLCs, cementoblasts, and osteoblasts. In contrast, Tβ4 activation using a Tβ4 peptide, promoted these processes by activation of Akt, p38, ERK MAPKs, and the NF-κB pathway. The expression of nuclear NFATc1 was upregulated by Tβ4 peptide in hPDLCs. Inhibition of the calcineurin/NFATc1 pathway by cyclosporin A and FK506, attenuated Tβ4-induced osteoblastic differentiation and activation of Wnt-related genes, as well as nuclear β-catenin in hPDLCs. In conclusion, this study demonstrates, for the first time, that Tβ4 is expressed in developing periodontal tissue and that its expression is associated with osteoblastic/cementoblastic differentiation. These results suggests that Tβ4 is a potential therapeutic target for periodontal regeneration or bone disease.

Melatonin promotes hepatic differentiation of human dental pulp stem cells: clinical implications for the prevention of liver fibrosis.

Melatonin's effect on hepatic differentiation of stem cells remains unclear. The aim of this study was to investigate the action of melatonin on hepatic differentiation as well as its related signaling pathways of human dental pulp stem cells (hDPSCs) and to examine the therapeutic effects of a combination of melatonin and hDPSC transplantation on carbon tetrachloride (CCl4 )-induced liver fibrosis in mice. In vitro hepatic differentiation was assessed by periodic acid-Schiff (PAS) staining and mRNA expression for hepatocyte markers. Liver fibrosis model was established by injecting 0.5mL/kg CCl4 followed by treatment with melatonin (5mg/kg, twice a week) and hDPSCs. In vivo therapeutic effects were evaluated by histopathology and by means of liver function tests including measurement of alanine transaminase (ALT), aspartate transaminase (AST), and ammonia levels. Melatonin promoted hepatic differentiation based on mRNA expression of differentiation markers and PAS-stained glycogen-laden cells. In addition, melatonin increased bone morphogenic protein (BMP)-2 expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist noggin. Furthermore, melatonin activated p38, extracellular signal-regulated kinase (ERK), and nuclear factor-κB (NF-κB) in hDPSCs. Melatonin-induced hepatic differentiation was attenuated by inhibitors of BMP, p38, ERK, and NF-κB. Compared to treatment of CCl4 -injured mice with either melatonin or hDPSC transplantation alone, the combination of melatonin and hDPSC significantly suppressed liver fibrosis and restored ALT, AST, and ammonia levels. For the first time, this study demonstrates that melatonin promotes hepatic differentiation of hDPSCs by modulating the BMP, p38, ERK, and NF-κB pathway. Combined treatment of grafted hDPSCs and melatonin could be a viable approach for the treatment of liver cirrhosis.

Effect of Wnt3a on Keratinocytes Utilizing in Vitro and Bioinformatics Analysis

Wingless-type (Wnt) signaling proteins participate in various cell developmental processes. A suppressive role of Wnt5a on keratinocyte growth has already been observed. However, the role of other Wnt proteins in proliferation and differentiation of keratinocytes remains unknown. Here, we investigated the effects of the Wnt ligand, Wnt3a, on proliferation and differentiation of keratinocytes. Keratinocytes from normal human skin were cultured and treated with recombinant Wnt3a alone or in combination with the inflammatory cytokine, tumor necrosis factor α (TNFα). Furthermore, using bioinformatics, we analyzed the biochemical parameters, molecular evolution, and protein–protein interaction network for the Wnt family. Application of recombinant Wnt3a showed an anti-proliferative effect on keratinocytes in a dose-dependent manner. After treatment with TNFα, Wnt3a still demonstrated an anti-proliferative effect on human keratinocytes. Exogenous treatment of Wnt3a was unable to alter mRNA expression of differentiation markers of keratinocytes, whereas an altered expression was observed in TNFα-stimulated keratinocytes. In silico phylogenetic, biochemical, and protein–protein interaction analysis showed several close relationships among the family members of the Wnt family. Moreover, a close phylogenetic and biochemical similarity was observed between Wnt3a and Wnt5a. Finally, we proposed a hypothetical mechanism to illustrate how the Wnt3a protein may inhibit the process of proliferation in keratinocytes, which would be useful for future researchers.

Osteoblastic differentiation of stromal ST-2 cells from octacalcium phosphate exposure via p38 signaling pathway

The present study investigated the role of mitogen-activated protein kinase (MAPK) signaling in osteoblastic differentiation of stromal ST-2 cells induced by synthetic octacalcium phosphate (OCP) incubation. Since our previous studies revealed that OCP consumes calcium ions in media during conversion to hydroxyapatite, the effect of the ions on ST-2 cell differentiation with or without OCP crystals was analyzed. The effect of presence or absence of MAPK inhibitors was also analyzed. OCP increased alkaline phosphatase (ALP) activity and the mRNA expression of differentiation markers via the p38 signaling pathway. The PD98059 MAPK inhibitor increased ALP activity and differentiation marker genes in cells cultured in OCP-coated wells. Reduction of calcium ions in the medium by EGTA increased the ALP activity without OCP in the presence of phosphate ion concentrations up to 7.5 mM. OCP may enhance osteoblastic differentiation through the p38 signaling pathway via the reduction of calcium ions induced by its physicochemical property.

The Role of Thymosin Beta 4 on Odontogenic Differentiation in Human Dental Pulp Cells

We recently reported that overexpression of thymosin beta-4 (Tβ4) in transgenic mice promotes abnormal hair growth and tooth development, but the role of Tβ4 in dental pulp regeneration was not completely understood. The aim of this study was to investigate the role of Tβ4 on odontoblastic differentiation and the underlying mechanism regulating pulp regeneration in human dental pulp cells (HDPCs). Our results demonstrate that mRNA and protein expression of Tβ4 is upregulated during odontogenic differentiation in HDPCs. Transfection with Tβ4 siRNA decreases OM-induced odontoblastic differentiation by decreasing alkaline phosphatase (ALP) activity, mRNA expression of differentiation markers, and calcium nodule formation. In contrast, Tβ4 activation with a Tβ4 peptide promotes these processes by enhancing the phosphorylation of p38, JNK, and ERK mitogen-activated protein kinases (MAPKs), bone morphogenetic protein (BMP) 2, BMP4, phosphorylation of Smad1/5/8 and Smad2/3, and expression of transcriptional factors such as Runx2 and Osterix, which were blocked by the BMP inhibitor noggin. The expression of integrin receptors α1, α2, α3, and β1 and downstream signaling molecules including phosphorylated focal adhesion kinase (p-FAK), p-paxillin, and integrin-linked kinase (ILK) were increased by Tβ4 peptide in HDPCs. ILK siRNA blocked Tβ4-induced odontoblastic differentiation and activation of the BMP and MAPK transcription factor pathways in HDPCs. In conclusion, this study demonstrates for the first time that Tβ4 plays a key role in odontoblastic differentiation of HDPCs and activation of Tβ4 could provide a novel mechanism for regenerative endodontics.

P.03.5 IN VIVO FORMATION OF 8-ISO-PROSTAGLANDIN F2A AND PLATELET ACTIVATION IN INFLAMMATORY BOWEL DISEASE

differentiation of human colonic fibroblasts and epithelial cells into myofibroblasts. Methods: Chronic colonic fibrosis was induced in C57BL/6 mice by successive 2.5% (w/v) DSS administration in drinking water for 6 weeks. After 6 weeks, the therapeutic effect of GED (30mg/kg/d), daily administered by oral gavage, was assessed macroscopically (weight/length of the colon, edema, ulcers, adhesions, thickness, dilatation), histologically (inflammatory infiltrate, collagen deposition) and biologically (colonic alpha-SMA, collagen I, TGFbeta1, CTGF, Smad3 and IL-13, by immunohistochemistry and immunoblotting). Differentiation of fibroblasts and intestinal epithelial cells (IECs) into myofibroblasts was induced by 4 day of TGFbeta administration (1 ng/mL and 10 ng/mL, respectively). Expression of aplha-SMA, fibronectine and the specific IECs markers (A33 and cytokeratin) was evaluated by quantitative RT-PCR. In vitro collagen deposition was observed by Picrosirius red staining. Results: In DSS-treated mice GED induced a significant 26% decrease of the colon weight/length ratio (p < 0.05) and ameliorated macroscopic and microscopic lesions determining 35% reduction of total macroscopic score (p < 0.05) and 91% (p < 0.01) of histological score. GED regulated the increased DSS-induced tissue expression of specific fibrotic markers, mainly Collagen I-III (48% p < 0.05), CTGF (35%, p < 0.05) and IL-13 (50%, p < 0.01). GED reduced mRNA expression of differentiation markers of fibroblasts and IECs, alpha-SMA (18%, p < 0.05 and 16%, p < 0.05, respectively) and fibronectine (45%, p < 0.005 and 67%, p < 0.005, respectively) and restored specific IECs markers. GED determined a 65% reduction of TGFbetainduced collagen deposition (p < 0.05), both in fibroblast and IECs. Conclusions: GED ameliorates the intestinal fibrosis in a DSS-induced chronic colitis in mice by controlling the main molecular and cellular mechanisms involved.

OC.07.1 MICRO-RNA EXPRESSION PROFILING IDENTIFIES MIR-29B AS A RELEVANT PRO-FIBROGENIC FACTOR IN CROHN'S DISEASE INTESTINAL STRICTURES

differentiation of human colonic fibroblasts and epithelial cells into myofibroblasts. Methods: Chronic colonic fibrosis was induced in C57BL/6 mice by successive 2.5% (w/v) DSS administration in drinking water for 6 weeks. After 6 weeks, the therapeutic effect of GED (30mg/kg/d), daily administered by oral gavage, was assessed macroscopically (weight/length of the colon, edema, ulcers, adhesions, thickness, dilatation), histologically (inflammatory infiltrate, collagen deposition) and biologically (colonic alpha-SMA, collagen I, TGFbeta1, CTGF, Smad3 and IL-13, by immunohistochemistry and immunoblotting). Differentiation of fibroblasts and intestinal epithelial cells (IECs) into myofibroblasts was induced by 4 day of TGFbeta administration (1 ng/mL and 10 ng/mL, respectively). Expression of aplha-SMA, fibronectine and the specific IECs markers (A33 and cytokeratin) was evaluated by quantitative RT-PCR. In vitro collagen deposition was observed by Picrosirius red staining. Results: In DSS-treated mice GED induced a significant 26% decrease of the colon weight/length ratio (p < 0.05) and ameliorated macroscopic and microscopic lesions determining 35% reduction of total macroscopic score (p < 0.05) and 91% (p < 0.01) of histological score. GED regulated the increased DSS-induced tissue expression of specific fibrotic markers, mainly Collagen I-III (48% p < 0.05), CTGF (35%, p < 0.05) and IL-13 (50%, p < 0.01). GED reduced mRNA expression of differentiation markers of fibroblasts and IECs, alpha-SMA (18%, p < 0.05 and 16%, p < 0.05, respectively) and fibronectine (45%, p < 0.005 and 67%, p < 0.005, respectively) and restored specific IECs markers. GED determined a 65% reduction of TGFbetainduced collagen deposition (p < 0.05), both in fibroblast and IECs. Conclusions: GED ameliorates the intestinal fibrosis in a DSS-induced chronic colitis in mice by controlling the main molecular and cellular mechanisms involved.

The Role of Heme Oxygenase-1 in the Proliferation and Odontoblastic Differentiation of Human Dental Pulp Cells

Introduction It was recently reported that heme oxygenase-1 (HO-1) activity is related to stem cell differentiation; however, the involvement of HO-1 in pulp cell growth and differentiation has not been well explored. The purpose of this study was to investigate the role of HO-1 in the growth and differentiation of human dental pulp cells (HDPCs). Methods We evaluated cell growth by MTT assay, mineralization by alizarin red staining, and differentiation marker mRNA expression by reverse transcriptase polymerase chain reaction. Results HO-1 induction by cobaltic protoporphyrin IX (CoPP) in HDPCs increased cell growth and mineralization and up-regulated the messenger RNA expression of such odontoblastic markers as alkaline phosphatase, osteopontin, bone sialoprotein, dentin matrix protein-1, and dentin sialophosphoprotein. Carbon monoxide scavenger, iron chelator, HO-1 inhibitor, and HO-1 small interfering RNA (siRNA) attenuated HDPC growth and differentiation. Conclusions CoPP treatment results in dental pulp cell proliferation and odontoblast differentiation that appears partly mediated by HO-1. Our results suggest that odontoblastic differentiation and growth are positively regulated by HO-1 induction and negatively regulated by HO-1 inhibition. Thus, pharmacologic HO-1 induction might represent a potent therapeutic approach for pulp capping and the regeneration of HDPCs.

Abstract LB-386: The role of neuronal connexins in the differentiation of neuroblastoma

Abstract Gap junctions are membrane channels allowing intercellular communication between adjacent cells, and regulate cellular processes such as metabolism, proliferation, and differentiation. Connexins (Cx), the constituent proteins of gap junctions, are aberrantly localized or have decreased expression in many cancers, and some connexins act as tumor suppressors when overexpressed. Connexins are also highly regulated during development and when overexpressed, some have been shown to enhance cellular differentiation. In the present study, the role of neuronal connexins, Cx36 and Cx45, in the differentiation of SH-SY5Y human neuroblastoma cells was investigated. Retinoic acid was used as the differentiating agent, as it is often employed in differentiation therapy of tumor cells. Retinoic acid induces neuritogenesis and expression of differentiation markers, such as growth associated protein 43 (Gap43), neuropeptide Y (NPY), and neuron specific enolase (NSE) in SH-SY5Y cells. Following 2, 4, and 10 days of retinoic acid induced differentiation of SH-SY5Y cells, Cx36 mRNA expression showed a significant increase compared to non-treated cells, while Cx45 mRNA expression remained stable. Cx36 was overexpressed using liposome-mediated transfection, and an increase in the mRNA levels of Gap43, NSE, and NPY was observed after 48 hours compared to control EGFP transfected cells. Cx36 overexpression also enhanced retinoic acid induced differentiation, as evidenced by the increase in mRNA expression of differentiation markers, Gap43 and NPY, following 2 days of treatment, and will now be confirmed following 4 and 10 days of treatment. Therefore, Cx36 overexpression induces and enhances the differentiation of SH-SY5Y neuroblastoma cells. Current studies involve knocking down Cx36 and Cx45 with shRNA and inducing differentiation of SH-SY5Y cells to determine whether connexin knock down diminishes neuritogenesis or differentiation marker expression. These studies provide insight into the role of connexins in the differentiation therapy approach to cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-386.

Frequency-Dependent Phenotype Modulation of Vascular Smooth Muscle Cells under Cyclic Mechanical Strain

Phenotype transformation of vascular smooth muscle cells (VSMCs) is known to be modulated by mechanical strain. The present study was designed to investigate how different frequencies of mechanical strain affected VSMC phenotype. VSMCs were subjected to the strains of 10% elongation at 0, 0.5, 1 and 2 Hz for 24 h using a Flexercell strain unit. VSMC phenotype was assessed by cell morphology, measurement of two-dimensional cell area, Western blotting for protein and RT-PCR for mRNA expression of differentiation markers. Possible protein kinases involved were evaluated by Western blotting with their specific antibodies. The strains at certain frequencies could induce a contractile morphology in VSMC with almost perpendicular alignment to the strain direction. The strains also regulated protein and mRNA expression of several differentiation markers, as well as the activation of extracellular signal-regulated kinases (ERKs), p38 MAP kinase and protein kinase B (Akt) in a frequency-dependent manner. Furthermore, the inhibition of the p38 pathway could block the frequency-induced phenotype modulation of VSMCs, but not inhibition of ERK or Akt pathways. These results indicate that the frequency of cyclic strain can result in the differentiated phenotype of VSMCs, and it is mediated at least partly by the activation of the p38 pathway.

Klotho Protein Promotes Adipocyte Differentiation

Mice with homozygous disruption of the klotho exhibit multiple age-related disorders and have barely detectable amounts of white adipose tissue. Although klotho expression in cultured adipocytes has been reported, little is known about its function in adipocytes. In the present study, we investigated the role of klotho on adipocyte differentiation. Adipocyte differentiation was induced by incubation of confluent 3T3-L1 cells with insulin, dexamethasone, and 1-methyl-3-isobutyl-xanthin. Klotho-siRNA and expression vector were produced for klotho suppression and overexpression, respectively. Klotho protein was purified for determination of the hormonal effect of klotho. Klotho mRNA and protein expression increased up to the 3rd d of differentiation. A peroxisome proliferator-activated receptor-gamma agonist increased klotho expression during the early period of adipocyte differentiation. The mRNA expression of adipocyte differentiation markers, such as CCAAT/enhancer-binding protein (C/EBP)alpha, C/EBPbeta, C/EBPdelta, peroxisome proliferator-activated receptor-gamma, and fatty acid binding protein 4, was decreased by klotho suppression, and increased 1.9- to 3.8-fold by klotho overexpression. The results of Oil Red O staining also suggested that klotho overexpression promoted adipocyte differentiation. Klotho protein stimulation resulted in a 2.4- to 4.6-fold increase in mRNA expression of differentiation markers compared with control, and the time course depended on adipocyte induction status. Western blot analysis showed that protein levels of C/EBPalpha and C/EBPdelta were increased by Klotho protein stimulation. These results suggest that klotho works as a hormonal factor to promote adipocyte differentiation in the early days, during the period of transient proliferation in the differentiation process, and that klotho may play an essential role in adipocyte differentiation.